Method of screening a midkine modulating agent

ABSTRACT

This invention relates to the discovery that midkine modulates pulmonary vasculature development and smooth muscle cell development. Modulation of midkine activity thus alters pulmonary vasculature development and smooth muscle cell development. The invention provides methods of modulating pulmonary disorders, smooth muscle cell related disorders, and pulmonary smooth muscle cell disorders. Disorders of particular interest include, but are not limited to, asthma and pulmonary hyperplasia. Further the invention relates to the discovery that TTF1 and HIF1 -α exhibit midkine modulating activity. The invention pertains to the discovery that midkine modulates myocardin activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and benefit of U.S. ProvisionalApplications: 60/519,473, filed on Nov. 12, 2003; 60/541,580, filed onFeb. 4, 2004; and 60/580,481, filed on Jun. 17, 2004; which are hereinincorporated by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to the field of modulation of pulmonary disorders,smooth muscle related disorders, and particularly pulmonary smoothmuscle disorders.

BACKGROUND OF THE INVENTION

Smooth muscle cells are a developmentally complex population. Smoothmuscle cells arise in multiple regions of the embryo from differentprecursor populations. For example, studies in chick/quail chimeras haveshown that unlike smooth muscle cells in the coronary arteries, smoothmuscle cells in the great vessels are derived from a subpopulation ofmesenchymal neural crest cells (Hood et al. (1992) Anat. Rec.234:291-300; Rosenquist et al. (1990) Ann. NY Acad. Sci. 588:106-119;Kirby et al. (1983) Science 220:1059-1061; Le Lievre et al (1975) J.Embryol. Exp. Morphol. 34:125-154; herein incorporated by reference intheir entirety.) Furthermore, the smooth muscle cells in most visceralorgans, including those of the respiratory system, are thought tooriginate from local mesenchyme (Cunha et al. (1992) Epith. Cell Biol.1:76-83, herein incorporated by reference).

Unlike skeletal and cardiac muscle cells, where cell differentiation isaccompanied by stable expression of muscle-specific genes (Weintraub etal. (1991) Science 251:761-766 and Olson et al. (1992) Genes & Dev.4:1454-1461, herein incorporated by reference in their entirety), smoothmuscle cells display remarkable phenotypic plasticity, and retain thecapacity to re-enter the cell cycle (Schwartz et al (1986) Circ. Res.58:427-444). This unusual characteristic of smooth muscle cellphenotypic modulation is often associated with the loss of many smoothmuscle cell-specific markers (Glukhova et al (1991) Am. J. Physiol.261:78-80; Frid et al (1992) Dev. Biol. 153:185-193; herein incorporatedby reference). Such alterations in smooth muscle cell proliferation anddifferentiation are associated with a variety of vascular diseasesincluding atherosclerosis, restenosis following angioplasty, andhypertension.

Smooth muscle proliferation is associated with numerous pulmonarydisorders such as asthma, airway hyperactivity, idiopathic pulmonaryhypertension, pulmonary hypertension, secondary pulmonary hypertension,COPD, and pulmonary hypotension. According to the Centers for DiseaseControl and Prevention, asthma affects almost six percent of Americanchildren and over six percent of American adults. Pulmonary hypertensionresulted in 7,139 deaths and 174,854 hospital visits in 1998. Currently,there is a low survival prognosis and no cure or simple treatment forpulmonary hypertension (Gerberding, J. (2003) Report to CongressPulmonary Hypertension Centers for Disease Control and Prevention,herein incorporated by reference). Numerous disorders including, but notlimited to emphysema, bronchitis, scleroderma, CREST, and congenitaldisorders, result in secondary pulmonary hypertension. Additionally, theN.I.H. reports that chronic obstructive pulmonary disorder is the fourthleading cause of death in the U.S. (N.I.H. Publication No. 03-5229,March 2003, herein incorporated by reference.)

Thus, there is a need in the art for a better understanding of themolecular mechanisms that regulate smooth muscle cell proliferation anddifferentiation, especially in respect to pulmonary disease. Developmentof methods of regulating smooth muscle proliferation is desirable.Development of methods of modulating pulmonary disorders is desirable.Development of methods of modulating pulmonary smooth muscle cellrelated disorders is particularly desirable.

SUMMARY OF THE INVENTION

Compositions and methods for diagnosis and therapy of disorders,particularly smooth muscle related disorders, pulmonary disorders,pulmonary smooth muscle related disorders, and pulmonary vascular smoothmuscle related disorders are provided. The invention encompasses methodsof modulating mammalian midkine activity, including modulating midkineexpression levels. In an aspect of the invention, pulmonary midkineactivity, including midkine expression levels, is modulated. Thecompositions and methods of the invention can be targeted towardpulmonary hypertension, chronic obstructive pulmonary disorder, asthma,and hypoxia related disorders.

In a first embodiment, the invention provides a method of preferentiallymodulating pulmonary tissue midkine expression levels, particularly in amammal. Transforming a mammalian cell with an isolated nucleic acidmolecule comprising an expression cassette with a pulmonarytissue-preferred promoter operably linked to a nucleotide sequence ofinterest directly or indirectly alters midkine expression levels inpulmonary tissue. Nucleotide sequences of interest include, but are notlimited to, midkine (SEQ ID NO:1), TTF-1 (SEQ ID NO:3), HIF-1 (SEQ IDNO:5), and fragments and variants thereof having midkine modulatingactivity or encoding a polypeptide having midkine modulating activity.Fragments and variants of a nucleotide sequence of interest includenucleotide sequences having at least 90% identity to a nucleotidesequence of interest, nucleotide sequences having at least 30 contiguousnucleotides of a nucleotide sequence of interest, nucleotide sequencesthat hybridize to a nucleotide sequence of interest under stringentconditions, and nucleotide sequences that complement a nucleotidesequence of interest. The method may be used to increase or decreasemidkine expression levels. In an embodiment, the transformed cell iscontained in an organism.

In a second embodiment, the invention provides a method of modulatingsmooth muscle development in a mammal by administering amidkine-modulating agent to the mammal. An aspect of smooth muscledevelopment is proliferation. Another aspect of smooth muscledevelopment is differentiation. In an aspect of the invention, the agentis administered to the pulmonary tissue of a mammal. In an aspect of theinvention the cell is contained in a mammal such as, but not limited to,a human, a mouse, a rat, a hamster, a rabbit, a dog, a pig, a goat, amonkey, a chimpanzee, and a cow. The midkine-modulating agent is notlimited to a particular mode of action but may bind midkine, bind amidkine receptor, or may alter midkine expression levels. In anotheraspect of the invention, the modulating agent includes, but is notlimited to a nucleic acid molecule, a polypeptide, a peptide, aglycoprotein, a transcription factor, an antibody, a small molecule, amidkine binding compound, or a HIF-1α modulating agent. An agent that isan isolated nucleic acid molecule comprises an expression cassettefurther comprising a promoter operably linked to a nucleotide sequenceof interest. Promoters useful in the invention include, but are notlimited to, inducible promoters, such as HIF-1 binding promoters,constitutive promoters; tissue-preferred promoters; pulmonary tissuepreferred promoters; and TTF-1 (thyroid specific transcription factor,also known as T-EBP, Ttf1, and Nkx2.1) binding promoters.

In a third embodiment, the invention provides a method of modulatingpulmonary smooth muscle development in a mammal by transforming apulmonary cell with an isolated nucleic acid molecule comprising anexpression cassette comprising a pulmonary tissue preferred promoteroperably linked to a nucleotide sequence of interest. An aspect ofsmooth muscle development is proliferation. Another aspect of smoothmuscle development is differentiation.

In a fourth embodiment, the invention provides a method of modulatingsmooth muscle cell related disorders by administering amidkine-modulating agent to a subject exhibiting a smooth muscle cellrelated disorder. In an aspect of the invention, the method ofmodulating the disorder decreases a smooth muscle cell related disorder.In another aspect, the method may be used to increase a smooth musclecell related disorder. In an aspect of the invention, the agent isadministered to pulmonary tissue.

In a fifth embodiment, the invention provides a method of modulating apulmonary disorder. The method comprises the step of transforming apulmonary cell with an isolated nucleic acid molecule comprising anexpression cassette comprising a pulmonary tissue-preferred promoteroperably linked to a nucleotide sequence of interest.

In a sixth embodiment, the invention provides a method of modulating apulmonary smooth muscle-cell related disorder. The method comprises thestep of transforming a pulmonary cell with an isolated nucleic acidmolecule comprising an expression cassette comprising a pulmonarytissue-preferred promoter operably linked to a nucleotide sequence ofinterest described elsewhere herein. Pulmonary smooth musclecell-related disorders include, but are not limited to asthma, airwayhyperactivity, and pulmonary hypertension. Pulmonary smooth muscle cellrelated disorders are further discussed elsewhere herein.

In a seventh embodiment, the invention provides a method of detecting amidkine pathway abnormality. The method involves obtaining a sample andassaying the midkine expression levels in the sample.

In an eighth embodiment, the invention provides a method of detecting apulmonary disorder. The method involves obtaining a pulmonary tissuesample and assaying the midkine expression levels in the sample.

In a ninth embodiment, the invention provides a method of screening fora midkine pathway abnormality. The method involves providing an isolatednucleic acid molecule comprising an expression cassette comprising amodified midkine promoter operably linked to a nucleotide sequenceencoding a reporter. In the invention, the promoter is a modifiedmidkine promoter having the nucleotide sequence set forth in SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16 orfragments or variants thereof capable of initiating transcription in amammalian cell. The invention comprises incubating the isolated nucleicacid molecule with a test sample and assaying the reporter. The reporterutilized in the invention can be any reporter known in the artincluding, but not limited to, luciferases, blue fluorescent proteins,green fluorescent proteins, CAT, GUS, β-galactosidases, and midkine. Inone aspect, the isolated nucleic acid molecule is transformed into acell. In an aspect of the invention, incubating the isolated nucleicacid molecule with a test sample occurs within a cell. In anotheraspect, incubating the nucleic acid molecule with a test sample occursin vitro. In an aspect of the invention, the test sample is selectedfrom the group including, but not limited to, cellular contents, celllysates, and cellular fractions.

In a tenth embodiment, the invention provides a method of screeningsubjects for a pulmonary disorder. The method involves providing anisolated nucleic acid molecule comprising an expression cassettecomprising a promoter operably linked to a nucleotide sequence encodinga reporter. In the invention, the promoter is a modified midkinepromoter having the nucleotide sequence set forth in SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16 or fragments orvariants thereof capable of initiating transcription in a mammaliancell. The invention comprises incubating the isolated nucleic acidmolecule with a pulmonary tissue sample and assaying the reporter.

In an eleventh embodiment, the invention provides a method of treating apulmonary disorder. The method comprises the step of administering atherapeutically effective amount of a midkine modulating agent to asubject exhibiting the disorder. In an aspect of the invention, thepulmonary disorder is a smooth muscle cell related disorder.

In a twelfth embodiment, the invention provides a method of treating apulmonary disorder comprising the step of transforming a cell with anisolated nucleic acid molecule comprising an expression cassettecomprising a pulmonary tissue-preferred promoter operably linked to anucleotide sequence of interest described elsewhere herein.

In a thirteenth embodiment, the invention provides a kit comprising anisolated nucleic acid molecule comprising an expression cassettecomprising a modified midkine promoter operably linked to a nucleotidesequence encoding a reporter.

In a fourteenth embodiment, the invention provides a transgenic mousecomprising at least one stably incorporated expression cassette in thegenome of at least one cell, said expression cassette comprising apromoter operably linked to a midkine nucleotide sequence. In an aspectof the invention, the promoter is an inducible, constitutive, ortissue-preferred promoter. The invention further provides transgenictissue and transgenic cells obtained from a transgenic mouse of theinvention, particularly pulmonary tissue and pulmonary cells. In anaspect of the invention, the mouse exhibits altered midkine expressionlevels. In an aspect of the invention, the mouse exhibits pulmonaryvasculature hypertrophy. In an aspect of the invention, the mouseexhibits a pulmonary disorder. In an additional aspect of the invention,the mouse exhibits an altered susceptibility to a pulmonary disorder. Inanother aspect, the invention provides a method of identifying midkinemodulating agents comprising providing a first and second transgeniccell, administering a compound of interest to the first cell, incubatingboth the first and second cells for a period of time, and monitoring thecells for a modulation of midkine activity.

In a fifteenth embodiment, the invention provides a method ofpreferentially modulating pulmonary tissue midkine expression levels,particularly in a mouse. The method involves providing a transgenicmouse comprising at least one stably incorporated expression cassette inthe genome of at least one cell, said expression cassette comprising aninducible promoter operably linked to a midkine nucleotide sequence. Inthe method, the transgenic midkine mouse is mated with a transgenicmouse comprising at least one stably incorporated expression cassette inthe genome of at least one cell, said expression cassette comprising apulmonary-tissue preferred promoter operably linked to a nucleotidesequence encoding an activator molecule. Double transgenic mice areobtained. In an aspect of the invention, a regulating compound such asbut not limited to, tetracycline or a tetracycline analog, isadministered to the double transgenic mice.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents the results of immunostaining for midkine in mouse lungtissue. The tissue in Panel A is from a TTF-1 null mouse embryo. Forcomparison, the tissue in Panel B is from a wild-type littermate embryo.In Panel A, the esophagus is indicated with an arrow and the vertebralcolumn is indicated with an arrowhead.

FIG. 2 depicts the results obtained from a midkine promoter activityassay. The expression constructs contained the midkine promoter (SEQ IDNO:7) operably linked to a luciferase reporter gene. The results inPanel A were obtained from JEG cells. JEG is a transformed humanplacental cell line with significantly reduced TTF-1 expression. Theresults in Panel B were obtained from H-441 cells. The H-441 cell lineis a human epithelial cell line expressing TTF-1. Increasing amounts (0to 500 ng) of a pCMV-TTF-1 expression plasmid were transfected into thecells.

FIG. 3 depicts the wild-type mouse midkine promoter (Panel A), a seriesof modified midkine promoter-reporter constructs (Panel B), and theresults obtained from promoter assays (Panel C). In panel A, circlesrepresent potential TTF-1 responsive elements (TREs), and starsrepresent consensus retinoic acid receptor sites. The modified midkinepromoter sequences represented by the diagram in panel B are provided asSEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10. The promoterswere operably linked to a luciferase reporter gene. The promoter assayspresented in panel C were performed in JEG cells in the presence (300)or absence (0) of pCMV-TTF-1. The assays were performed using the fulllength midkine promoter (SEQ ID NO:7), and the deletion constructs (2.0kb SEQ ID NO:8, 1.7 kb SEQ ID NO:9, and 1.0 kb SEQ ID NO:10).

FIG. 4 depicts the wild-type mouse midkine promoter (Panel A), a seriesof modified midkine promoter constructs (Panel B), and the resultsobtained from promoter assays (Panel C). In panel A, circles representpotential TTF-1 responsive elements (TREs), and stars representconsensus retinoic acid receptor sites. The modified midkine promotersequences represented by the diagram in panel B are provided as SEQ IDNO:11 (a), SEQ ID NO:12 (b), SEQ ID NO:13 (c), and SEQ ID NO:14 (d). Thepromoters were operably linked to a luciferase reporter gene. Thepromoter assays presented in panel C were performed in JEG cells in thepresence (300) or absence (0) of pCMV-TTF-1. The assays were performedusing the full length midkine promoter (SEQ ID NO:7), and site directedmutation constructs (SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQID NO:14).

FIG. 5, panel A depicts a schematic of the pulmonary tissue preferredmidkine expression system used in the transgenic mice of the invention.The box labeled hSPC indicates the human surfactant protein C promoteroperably linked to the reverse tetracycline transactivator cDNA (rtTA,striped box). rtTA molecules are indicated by rectangles overlayed withtriangles. Tetracycline-like molecules are indicated by speckledtriangles (Dox). Activated rtTA molecules are indicated by a combinationof the rtTA shapes and tetracycline-like molecule speckled triangles.The tetO-MK transgene consists of the murine midkine cDNA (SEQ ID NO:25,white box) operably linked to the minimal cytomegalovirus (CMV) promotercontaining seven concatemerized tetracycline operon binding sites(rectangles overlayed with speckled triangles). Midkine molecules aredepicted as white ovals labeled MK. FIG. 5, panel B depicts resultsobtained from RT-PCR reactions performed on 2 μg total RNA isolated fromdouble transgenic (SP-C-rtTA^(+/tg), (tetO)₇-CMV-MK^(+/tg or tg/tg)) andcontrol littermates in the presence (+Dox) or absence (−Dox) ofdoxycycline.

FIG. 6 presents histology and immunohistochemical results from pulmonarytissue of double transgenic mice and wild type mice for comparison.Tissue from wild-type mice (Non-transgenic) is presented in the rightcolumn; tissue from SP-C-rtTa^(+/tg), (tetO)₇-CMV-MK^(+/tg or tg/tg)mice (Double-transgenic) is presented in the left column. The tissuesections in panels A and B were stained with hematoxylin and eosin. Thetissue sections in panels C and D were incubated with anti-midkineantibodies (anti-MK). The tissue sections in panels E and F wereincubated with anti-α smooth muscle actin antibodies (anti-α-SMA). Thetissue sections in panels G and H were incubated with anti-Caldesmonantibodies (anti-Caldesmon). The bar indicates 50 μm.

FIG. 7 presents average right ventricle hypertrophy (RVH), distalpulmonary vessel counts (No. distal vessels), and percent distalmuscularization of mice in normoxic and hypoxic conditions. Forcomparison the results obtained from wild-type (control) and doubletransgenic mice (SP-C-rtTA^(+/tg), (tetO)₇-CMV-MK^(+/tg or tg/tg)) arepresented. The standard error values follow the means.

FIG. 8 presents immunostaining of bronchioles and bronchial smoothmuscle obtained from human subjects exhibiting a COPD (emphysema). Thesections were incubated with anti-midkine antibodies. Panel F was notincubated with primary antibodies.

FIG. 9 presents immunohistochemical results from pulmonary tissue ofFVB/N and CAST/eiJ mice. Panel A and Panel B present midkine staining ofFVB/N pulmonary tissue after exposure to normoxia (A) and hypoxia (B)for five weeks. Panel C and Panel D present midkine staining of CAST/eiJpulmonary tissue after exposure to normoxia (C) and hypoxia (D) for fiveweeks. Panels E and F present midkine (E) and α-SMA (F) staining ofCAST/eiJ pulmonary tissue after exposure to hypoxia for four weeks.Panels G, H, I, J, K, and L present midkine staining of CAST/eiJpulmonary tissue after exposure to hypoxia for 1 day (G), 3 days (H), 1week (I), 2 weeks (J), 3 weeks (K), and 4 weeks (L). Images are at 40×(Panels A-D, G-L) and 100× (Panels E-F). The bar indicates 100 μM.

FIG. 10 presents the results obtained from PCR performed on cDNAgenerated by reverse transcription of RNA isolated from JEG-3 (Lane 2),MFLM-4 (Lane 4), and H-441 (Lane 6) cells after exposure to hypoxia. Forcomparison RT-PCR was performed on cDNA generated by reversetranscription of RNA isolated from JEG-3 (Lane 1), MFLM-4 (Lane 3), andH-441 (Lane 5) cells maintained in normoxia. Results of the PCRreactions containing HIF-1α primers, midkine primers (MK), and GAPDHprimers are indicated. The graph indicates the fold change in midkineexpression.

FIG. 11 depicts a schematic of the wild-type midkine promoter and theresults of multiple promoter assays assessing the effects of HIF-1α andTTF-1 on the midkine promoter (SEQ ID NO:7) and modified midkinepromoters. The promoters were operably linked to a luciferase reportergene. Panel A presents the wild-type midkine promoter (SEQ ID NO:7).TTF-1 response elements (TRE) are indicated by empty boxes; HIF-1αresponse elements (HRE) are indicated by solid boxes. In panel B, thewild-type promoter construct and the indicated amount of pCMV-HIF-1α orpCMV-TTF1 were transformed into JEG cells, and promoter assays wereperformed. Results are indicated in luciferase units X 10⁵/β-gal or10⁴/β-gal as indicated. Panel C presents the results of promoter assaysperformed with the wild-type midkine promoter or a truncated midkinepromoter (SEQ ID NO:9). Assays were performed in the presence or absenceof 320 ng of pCMV-HIF-1α. Panel D presents the results of promoterassays performed with the wild-type midkine promoter or site directedmodified midkine promoters (SEQ ID NO:15 and SEQ ID NO:16). Assays wereperformed in the presence or absence of 320 ng of pCMV-HIF-1α.

FIG. 12 presents micrographs of tissue sections from double-transgenicand non-transgenic mice stained with α-SMA specific antibodies. Smallblood vessels are indicated with arrows. Large blood vessels areindicated with arrowheads. The tissue in panels A and C were obtainedfrom double-transgenic mice. The tissue in panels B and D were obtainedfrom non-transgenic littermates. Panels A and B present tissue fromanimals fed doxycycline from conception to post-natal day 21. Panels Cand D present tissue from animals fed doxycycline from post-natal day 21to 9 weeks of age. The bar indicates 50 μm.

FIG. 13 presents results of peripheral pulmonary arterial patternassessments. Panel A presents pulmonary arteriograms performed on adultnon-transgenic (top row) and double transgenic (bottom row) mice. PanelB presents hematoxylin and eosin stained pulmonary tissue sectionsobtained from non-transgenic and double transgenic mice. The barindicates 100 μm. Panel C presents a summary of pulmonary arterialdensity in non-transgenic (NTG) and double transgenic (MK-TG) mice. N=8.Panel D presents the results of Western blot analysis of Tie-2 andβ-actin expression in whole lung homogenates from non-transgenic(Non-TG) and double transgenic (double-TG) mice.

FIG. 14, panel A presents results obtained from RT-PCR analysis ofmyocardin, calponin, SM-22, α-SMA, and GAPDH expression in MFLM-4 cellsincubated with a placebo (−MK) or with midkine (+MK). Panel B presentsresults of real time RT-PCR analysis of myocardin expression relative toβ-actin in lungs of transgenic (MK-TG) and non-transgenic mice (NTG).Panel C presents the myocardin/β-actin expression ratio in the lungs ofCAST/eiJ mice exposed to hypoxia for the indicated number of weeks.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of modulating midkine activity,midkine expression levels, and pulmonary midkine expression levels. Theinvention further provides methods of modulating smooth muscle celldevelopment, particularly pulmonary smooth muscle cell development, moreparticularly pulmonary vascular smooth muscle cell development.Additionally the invention provides methods of modulating smooth musclecell related disorders, pulmonary disorders, pulmonary smooth musclecell related disorders, and pulmonary vascular smooth muscle cellrelated disorders. The invention provides methods of detecting midkinepathway abnormalities and pulmonary disorders. Further the inventionprovides methods of screening for a midkine pathway abnormality and fora pulmonary disorder. The invention provides methods of treating apulmonary disorder. In addition, the invention provides methods ofidentifying a midkine modulating agent, smooth muscle cell developmentmodulating agents, and pulmonary disorder modulating agents.Compositions of the invention include transgenic mice expressingmidkine, particularly mice expressing midkine in an inducible, pulmonarytissue-preferred manner. Expression of midkine may alter a transgenicanimal's susceptibility to smooth muscle cell related disorders or topulmonary disorders, particularly pulmonary smooth muscle cell relateddisorders. Additionally the invention provides modified midkinepromoters, and kits comprising a modified midkine promoter.

The invention relates to compositions and methods drawn to the murinemidkine promoter, modified midkine promoters, and transgenic animalscomprising a midkine transgene. The promoter sequences are useful forscreening for a midkine pathway abnormality, screening subjects for apulmonary disorder, evaluating compounds for midkine modulatingactivity, evaluating compounds for effects on smooth muscle celldevelopment, and evaluating compounds for pulmonary disorder modulatingactivity. Methods of modulating midkine activity, modulating smoothmuscle cell development, pulmonary development, pulmonary smooth muscledevelopment, smooth muscle cell related disorders, pulmonary disorders,and pulmonary smooth muscle cell disorders involve administering amidkine modulating agent to a cell or subject. Particularly, theinvention provides methods of modulating midkine expression that involveadministering a midkine modulating agent.

A “midkine modulating agent” is a compound that modulates a midkineactivity. Modulation may be an increase or decrease in a midkineactivity. A midkine modulating compound will modulate a midkine activityby at least 1%, 5%, preferably 10%, 20%, more preferably 30%, 40%, 50%,60%, yet more preferably 70%, 80%, 90%, or 100% as compared to anuntreated or placebo treatment effect. By “midkine activity” is intendedany activity exhibited by the wild-type murine midkine described herein.Such activities include, but are not limited to, midkine expression,immunogenicity, midkine receptor binding, heparin binding, modulation ofcell differentiation, modulation of cell proliferation, protein tyrosinephosphatase binding, nucleolin binding, retinoic acid responsiveness,immunoreactivity, growth enhancement, neurite outgrowth, chemotacticactivity, mitogenesis, angiogenesis modulation, neuron survivalmodulation, fibrinolysis, myocardin modulation, cell growth, cellmigration, and tumorigenesis (Muramatsu et al. (1993) Dev. Biol.159:392-402; Choundhri et al. (1997) Cancer Res. 57:1814-1819; Owada etal. (1999) J. Neurochem 73:2084-2092; Kojima et al. (1995) J. Biol.Chem. 270:9590-9596; Muramatsu & Muramatsu (1991) Biochem. Biophys. Res.Commun. 177:652-658; Kurtz et al. (1995) Crit. Rev. Oncol. 6:151-177;Tsutsui et al. (1993) Cancer Res. 53:1281-1285; Nakagawara et al. (1995)Cancer Res. 55:1792-1797; Garver et al. (1993) Am J. Respir. Cell Biol.9:463-466; O'Brien et al. (1996) Cancer Res. 56:2515-2518; Kaname et al.(1996) Biochem. Biophys. Res. Commun. 219:256-260; Miyashiro et al.(1996) Cancer Lett 106:287-291; Miyashiro et al. (1997) Breast CancerRes. Treat. 43:1-6; Takada et al. (1997) J. Biochem. 122:453-458; Maedaet al (1999) J. Biol. Chem. 274:12474-12479; Horiba et al. (2000) J.Clin. Invest. 105:489-495; Aridome et al. (1998) Br. J Cancer78:472-477; Walker, John, ed. (2002) Protein Protocols on CD-ROM v. 2;and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology,(Greene Publishing and Wiley-Interscience, New York; U.S. patentapplication Nos. 20020034768 and 20030053989; herein incorporated byreference in their entirety). Modulation of midkine activity includesbut is not limited to modulation of a midkine activity such as proteintyrosine phosphatase binding or modulation of midkine expression levels.Thus, modulation of midkine expression is an alteration of a midkineactivity. Methods for assaying midkine activity are known in the art andare described elsewhere herein.

In an embodiment, the invention provides compositions with an alteredmidkine activity and methods of modulating midkine activity. By “alteredmidkine activity” is intended a change of at least 1%, 5%, preferably10%, 20%, more preferably 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% or morein a midkine activity. The change may be an increase or a decrease inthe activity. Further, a compound that increases one midkine activitymay decrease a different midkine activity.

It is understood that any midkine activity assay can be used to assaymidkine modulating activity including, but not limited to, expressionassays, immunogenicity, myocardin interaction assays, myocardinactivation assays, receptor binding assays, heparin binding assays, celldifferentiation assays, cell adhesion assays, FACS analysis, proteintyrosine phosphatase binding assays, growth assays, chemotactic assay,Western blots, EM, SEM, light microscopy, retinoic acid responsivenessassays, nucleolin binding assays, size exclusion chromatography, andcapture ELISAs with multiple midkine antibodies. (Reynolds etal. (2004)J. Biol. Chem. 279: 37124-37132; Reynolds et al. (2003) Dev. Dyn.227:227-237; Muramatsu et al. (1993) Dev. Biol. 159:392-402; Choundhriet al. (1997) Cancer Res. 57:1814-1819; Owada et al. (1999) J. Neurochem73:2084-2092; Kojima et al. (1995) J. Biol. Chem. 270:9590-9596;Muramatsu & Muramatsu (1991) Biochem. Biophys. Res. Commun. 177:652-658;Kurtz et al. (1995) Crit. Rev. Oncol. 6:151-177; Tsutsui et al. (1993)Cancer Res. 53:1281-1285; Nakagawara et al. (1995) Cancer Res.55:1792-1797; Garver et al. (1993) Am J. Respir. Cell Biol. 9:463-466;O'Brien et al. (1996) Cancer Res. 56:2515-2518; Kaname et al. (1996)Biochem. Biophys. Res. Commun. 219:256-260; Miyashiro et al. (1996)Cancer Lett 106:287-291; Miyashiro et al. (1997) Breast Cancer Res.Treat. 43:1-6; Takada et al. (1997) J. Biochem. 122:453-458; Maeda et al(1999) J. Biol. Chem. 274:12474-12479; Horiba et al. (2000) J. Clin.Invest. 105:489-495; Aridome et al. (1998) Br. J Cancer 78:472-477;Walker, John, ed. (2002) Protein Protocols on CD-ROM v. 2; and Ausubelet al., eds. (1995) Current Protocols in Molecular Biology, (GreenePublishing and Wiley-Interscience, New York; U.S. patent applicationNos. 20020034768 and 20030053989; herein incorporated by reference intheir entirety).

A “HIF-1α modulating agent” is a compound that modulates a HIF-1αactivity such as altering midkine activity, modulating the HIF-1α/βinteraction, oxygen level responsiveness, DNA binding, nucleartranslocation, and degradation susceptibility. A HIF-1α modulatingcompound will modulate a HIF-1α activity by at least 1%, 5%, preferably10%, 20%, more preferably 30%, 40%, 50%, 60%, yet more preferably 70%,80%, 90%, or 100% as compared to an untreated or placebo treatmenteffect. By “HIF-1α activity” is intended any activity exhibited by thewild-type murine HIF-1α. Such activities include, but are not limitedto, HIF-1α expression, interacting with HIF-1β, binding DNA, cellulartranslocation, degradation susceptibility, and midkine stimulation.Thus, modulation of HIF-1α expression is an alteration of HIF-1αactivity. Methods for assaying HIF-1α activity are known in the art andare described elsewhere herein.

In an embodiment, the invention provides compositions with an alteredHIF-1α activity and methods of modulating HIF-1α activity. By “alteredHIF-1α activity” is intended a change of at least 1%, 5%, preferably10%, 20%, more preferably 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% or morein a HIF-1α activity. The change may be an increase or a decrease in theactivity. Further, a compound that increases one HIF-1α activity maydecrease a different HIF-1α activity.

It is understood that any HIF-1α activity assay can be used to assayHIF-1α modulating activity including, but not limited to, expressionassays, DNA binding assays, HIF-1α/β binding assays, HIF-1α/βdimerization assays, Western blots, EM, SEM, light microscopy, DAPIstaining, and translocation assays. (Walker, John, ed. (2002) ProteinProtocols on CD-ROM v. 2; and Ausubel et al., eds. (1995) CurrentProtocols in Molecular Biology, (Greene Publishing andWiley-Interscience, New York).

An embodiment of the invention provides methods of modulating myocardinactivity. In the methods modulating midkine activity modulates myocardinactivity. By “myocardin activity” is intended any activity associatedwith murine myocardin including, but not limited to, smooth muscle generegulation (Veyssier-Belot & Cacoub (1999) Cardiovascular Research44:274-282), serum response factor interaction, myogenic activity,modulation of α-SMA, modulation of calponin, immunogenicity, andmodulation of SM-22. Methods of detecting myocardin activity are knownin the art; any method of assaying myocardin activity may be used in themethods of the invention.

In an embodiment, the invention provides a method of altering expressionof a native midkine nucleotide sequence in an animal, particularly ofmodulating midkine expression in a tissue preferred manner. In theembodiment a midkine modulating agent comprising an expression cassettecomprising a promoter operably linked to a nucleotide sequence havingmidkine modulating activity or encoding a polypeptide having midkinemodulating activity is administered. In an embodiment the promoter is atissue preferred promoter such as, but not limited to, a pulmonarytissue preferred promoter or a smooth muscle cell preferred promoter. Inan embodiment, the midkine modulating agent alters the HIF-1α/βinteraction.

By “alters midkine expression levels” is intended that the expression ofthe midkine nucleotide sequence in a transgenic cell, transgenic tissue,or transgenic cell or tissue of a transgenic animal of the inventiondiffers from expression levels in a non-transgenic cell, non-transgenictissue, or a non-transgenic animal by at least 1%, 2%, 3%, 4%, 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100%. The difference may be an increase or decrease inexpression levels.

Methods of assaying expression levels are known in the art and include,but are not limited to, qualitative Western blot analysis,immunoprecipitation, radiological assays, polypeptide purification,spectrophotometric analysis, Coomassie staining of acrylamide gels,ELISAs, RT-PCR, 2-D gel electrophoresis, microarray analysis, in situhybridization, chemiluminescence, silver staining, enzymatic assays,ponceau S staining, multiplex RT-PCR, immunohistochemical assays,radioimmunoassay, colorimetric analysis, immunoradiometric assays,immunochemistry, positron emission tomography, Northern blotting,fluorometric assays, fluorescence activated cell sorter staining ofpermeabilized cells, radioimmunosorbent assays, real-time PCR,hybridization assays, sandwich immunoassays, flow cytometry, SAGE,differential amplification, or electronic analysis. See, for example,Ausubel et al, eds. (2002) Current Protocols in Molecular Biology,Wiley-Interscience, New York, N.Y.; Coligan et al (2002) CurrentProtocols in Protein Science, Wiley-Interscience, New York, N.Y.; Sun etal. (2001) Gene Ther. 8:1572-1579; de Jager et al. (2003). Clin. & Diag.Lab. Immun. 10:133-139; U.S. Pat. Nos. 6,489,4555; 6,551,784; 6,607,879;4,981,783; and 5,569,584; herein incorporated by reference in theirentirety.

An embodiment of the invention is a method of detecting a midkinepathway abnormality. The method comprises obtaining a sample andassaying the midkine expression level in the sample. An increase ordecrease in expression level compared to standard midkine expressionlevels in a similar sample obtained from a healthy subject, eitherdirectly or indirectly (for example, a predetermined standard) indicatesa midkine pathway abnormality. By “midkine pathway” is intended anypathway that impacts midkine expression, directly or indirectly. A“midkine pathway abnormality” is an alteration in any stage of a midkinepathway that alters midkine expression. Assays for a midkine pathwayabnormality include but are not limited to midkine expression assays andmidkine promoter expression assays.

Another embodiment provides a method of detecting a pulmonary disorder.The method comprises obtaining a pulmonary tissue sample and assayingthe midkine expression levels in the pulmonary tissue sample. By“pulmonary tissue” is intended any tissue obtained from the lungs,including but not limited to, the lungs, bronchi, bronchioles, alveoli,and developmentally related tissues. A tissue is one or more relatedcells. By “pulmonary smooth muscle cell” is intended any smooth musclecell associated with pulmonary tissue including, but not limited to,smooth muscle cells associated with bronchial conducting airways andpulmonary arterial cells. By “pulmonary vascular smooth muscle cell” isintended any smooth muscle cell associated with the pulmonaryvasculature including, but not limited to, pulmonary vascular cells andtheir precursors.

Midkine modulating agents include, but are not limited to, midkine,TTF-1, HIF1-α, oxygen, siRNA, anti-sense RNA, midkine binding compounds,midkine receptor binding compounds, and compounds that alter midkineexpression such as a HIF 1-α modulating agent. Additional midkinemodulating agents may be identified using the methods of the invention.

In several embodiments of the invention, the midkine modulating agent isan isolated nucleic acid molecule comprising an expression cassette. Theexpression cassette comprises a promoter operably linked to a nucleotidesequence of interest. Nucleotide sequences of interest exhibit a midkinemodulating activity or encode a polypeptide having midkine modulatingactivity. Nucleotide sequences of interest include, but are not limitedto, midkine (SEQ ID NO:1; SEQ ID NO:25), thyroid transcription factor-1(TTF-1; SEQ ID NO:3), and hypoxia inducing factor-1 (HIF-1α; SEQ ID NO:5), and fragments and variants thereof.

Fragments and variants of the nucleotide sequences of interest andpolypeptides encoded thereby are also encompassed by the presentinvention. Fragments and variants of the modified midkine promoters(discussed elsewhere herein) are also encompassed by the presentinvention. By “fragment” is intended a portion of the nucleotidesequence or a portion of the amino acid sequence and hence proteinencoded thereby. Fragments of a nucleotide sequence may encode proteinfragments that retain the biological activity of the native protein andhence exhibit a midkine activity. Fragments of a promoter nucleotidesequence may retain biological activity and drive expression.Alternatively, fragments of a nucleotide sequence that are useful ashybridization probes generally do not retain biological activity. Thus,fragments of a nucleotide sequence may range from at least about 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, up to881 nucleotides for SEQ ID NO:1; from at least about 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000,1050, 1100, up to about 1119 nucleotides for SEQ ID NO:3; from at leastabout 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750,800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350,1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950,2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550,2600, 2650, 2700, 2750, 2800, 2850, 2900, 2950, 3000, 3050, 3100, 3150,3200, 3250, 3300, 3350, 3400, 3450, 3500, 3550, 3600, 3650, 3700, 3750,3800, 3850, 3900, 3950, up to about 3973 nucleotides for SEQ ID NO:5;from at least about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250,1300,1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850,1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450,2500, 2550, up to about 2559 nucleotides for SEQ ID NO:7, 11, 12, 13, or14; from at least about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250,1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850,1900, 1950, 2000, 2050, up to about 2074 nucleotides for SEQ ID NO:8,from at least about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300,1350, 1400, 1450, 1500, 1550, 1600, 1650,up to about 1677 nucleotidesfor SEQ ID NO:9; from at least about 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, up to about 1037nucleotides for SEQ ID NO:10, from at least about 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000,1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600,1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200,2250, 2300, 2350, 2400, 2450, 2500, 2550, up to about 2566 nucleotidesfor SEQ ID NO:15 or 16; from at least about 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, up to 695 nucleotides for SEQ ID NO:25.

A fragment of a nucleotide sequence of interest that encodes abiologically active portion of a polypeptide of interest will encode atleast 15, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, or 140contiguous amino acids, or up to the total number of amino acids presentin the full-length protein. Fragments of a nucleotide sequence orinterest that are useful as hybridization probes or PCR primersgenerally need not encode a biologically active portion of a protein.

Thus, a fragment of a nucleotide sequence of interest may encode abiologically active portion of midkine, a midkine promoter, or a midkinemodulating agent or it may be a fragment that can be used as ahybridization probe or PCR primer using methods disclosed below. Abiologically active portion of a midkine can be prepared by isolating aportion of one of the midkine nucleotide sequences of the invention,expressing the encoded portion of the midkine protein (e.g., byrecombinant expression in vitro), and assessing the activity of theencoded portion of the midkine protein. Nucleic acid molecules that arefragments of a midkine nucleotide sequence comprise at least about 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150,200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, upto 881 nucleotides for SEQ ID NO:1 or at least about 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, up to 695 nucleotides for SEQ IDNO:25. A biologically active portion of a midkine promoter can beprepared by isolating a portion of the promoter nucleotide sequencedisclosed herein, and assessing the activity of the portion of thepromoter. Nucleic acid molecules that are fragments of a midkinepromoter comprise from at least about 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450,500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100,1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700,1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300,2350, 2400, 2450, 2500, 2550, up to about 2559 nucleotides for SEQ IDNO:7, 11, 12, 13, or 14; from at least about 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100,1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700,1750, 1800, 1850, 1900, 1950, 2000, 2050, up to about 2074 nucleotidesfor SEQ ID NO:8, from at least about 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150,1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, up to about1677 nucleotides for SEQ ID NO:9; from at least about 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000,up to about 1037 nucleotides for SEQ ID NO:10, from at least about 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150,200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450,1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050,2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550, up to about2566 nucleotides for SEQ ID NO:15 or 16.

By “variants” is intended substantially similar sequences. Fornucleotide sequences, conservative variants include those sequencesthat, because of the degeneracy of the genetic code, encode the aminoacid sequence of one of the midkine polypeptides of the invention.Naturally occurring allelic variants such as these can be identifiedwith the use of well-known molecular biology techniques, as, forexample, with polymerase chain reaction (PCR) and hybridizationtechniques as outlined below. Variant nucleotide sequences also includesynthetically derived nucleotide sequences, such as those generated, forexample, by using site-directed mutagenesis [but which still encodemidkine]. Generally, variants of a particular nucleotide sequence ofinterest will have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, and more preferably at least about 98%, 99% or more sequenceidentity to that particular nucleotide sequence as determined bysequence alignment programs described elsewhere herein using defaultparameters.

By “variant” protein is intended a protein derived from the nativeprotein by deletion (so-called truncation) or addition of one or moreamino acids to the N-termial and/or C-terminal end of the nativeprotein; deletion or addition of one or more amino acids at one or moresites in the native protein; or substitution of one or more amino acidsat one or more sites in the native protein. Variant proteins encompassedby the present invention are biologically active, that is they continueto possess the desired biological activity of the native protein, thatis, a midkine modulating activity as described herein. Such variants mayresult from, for example, genetic polymorphism or from humanmanipulation. Biologically active variants of a native midkinemodulating protein of the invention will have at least about 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, and more preferably at least about 98%,99% or more sequence identity to the amino acid sequence for the nativeprotein as determined by sequence alignment programs described elsewhereherein using default parameters. A biologically active variant of aprotein of the invention may differ from that protein by as few as 1-15amino acid residues, as few as 1-10, such as 6-10, as few as 5, as fewas 4, 3, 2, or even 1 amino acid residue.

The proteins of the invention may be altered in various ways includingamino acid substitutions, deletions, truncations, and insertions.Methods for such manipulations are generally known in the art. Forexample, amino acid sequence variants of the midkine modulatingpolypeptides can be prepared by mutations in the DNA. Methods formutagenesis and nucleotide sequence alterations are well known in theart. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S.Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques inMolecular Biology (MacMillan Publishing Company, New York) and thereferences cited therein. Guidance as to appropriate amino acidsubstitutions that do not affect biological activity of the protein ofinterest may be found in the model of Dayhoff et al. (1978) Atlas ofprotein Sequence and Structure (Natl. Biomed. Res. Found., Washington,D.C.), herein incorporated by reference. Conservative substitutions,such as exchanging one amino acid with another having similarproperties, may be preferable.

Thus, the genes and nucleotide sequences of the invention include boththe naturally occurring sequences as well as mutant forms. Likewise, theproteins of the invention encompass both naturally occurring proteins aswell as variations and modified forms thereof. Such variants willcontinue to possess the midkine modulating activity. Obviously, themutations that will be made in the DNA encoding the variant must notplace the sequence out of reading frame and preferably will not createcomplementary regions that could produce secondary mRNA structure. See,EP Patent Application Publication No. 75,444.

When it is difficult to predict the exact effect of the substitution,deletion, or insertion in advance of doing so, one skilled in the artwill appreciate that the effect will be evaluated by routine screeningassays. That is, the activity can be evaluated by suitable assays suchas, but not limited to, promoter activity assays or midkine activityassays such as immunogenic activity.

Variant nucleotide sequences also encompass sequences derived from amutagenic and recombinogenic procedure such as DNA shuffling. With sucha procedure, one or more different promoter sequences or nucleotidesequences of interest can be manipulated to create a new sequencepossessing the desired properties. In this manner, libraries ofrecombinant polynucleotides are generated from a population of relatedsequence polynucleotides comprising sequence regions that havesubstantial sequence identity and can be homologously recombined invitro or in vivo. For example, using this approach, sequence motifsencoding a domain of interest may be shuffled between the modifiedmidkine promoters of the invention and other known promoters to obtain anew promoter with an altered property of interest e.g. alteredexpression levels or altered midkine activity. Strategies for such DNAshuffling are known in the art. See, for example, Stemmer (1994) Proc.Natl. Acad. Sci. 91:10747-10751; Stemmer (1994) Nature 370:389-391;Crameri et al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J.Mol. Biol. 272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci.94:4504-4509; Crameri et al. (1998) Nature 391:288-291; Miyazaki (2002)Nucleic Acids Research 30:E139-9; Song et al. (2002) Appl. Environ.Microbiol. 68:6146-51; Hayes et al. (2002) Proc. Natl Acad. Sci.99:15926-31; Coco et al. (2001) Nature Biotechnol. 19:354-9; Kikuchi etal. (2000) Gene 243:133-7; and U.S. Pat. Nos. 5,606,793 and 5,837,458.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, (d)“percentage of sequence identity”, and (e) “substantial identity”.

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence or the complete cDNA or gene sequence.

(b) As used herein “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e. gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. Generally the comparison window is at least 20 contiguousnucleotides in length, and optionally can be 30, 40, 50, 100, or longer.Those of skill in the art understand that to avoid a high similarity toa reference sequence due to inclusion of gaps in the polynucleotidesequence a gap penalty is typically introduced and is subtracted fromthe number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent sequence identity between anytwo sequences can be accomplished using a mathematical algorithm.Preferred, non-limiting examples of such mathematical algorithms are thealgorithm of Myers and Miller (1988) CABIOS 4:11-17; the local homologyalgorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the homologyalignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol.48:443-453; the search-for-similarity-method of Pearson and Lipman(1988) Proc. Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin andAltschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as inKarlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity. Forpurposes of the present invention, comparison of nucleotide or proteinsequences for determination of percent sequence identity to thesequences disclosed herein is preferably made using the GCG program GAP(Version 10.00 or later) with its default parameters or any equivalentprogram. By “equivalent program” is intended any sequence comparisonprogram that, for any two sequences in question, generates an alignmenthaving identical nucleotide or amino acid residue matches and anidentical percent sequence identity when compared to the correspondingalignment generated by the preferred program. Alignment may also beperformed manually by inspection.

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid or polypeptide sequences makes reference to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

(e)(i) The term “substantial identity” of polynucleotide sequences meansthat a polynucleotide comprises a sequence that has at least 70%sequence identity, preferably at least 80%, more preferably at least90%, and most preferably at least 95%, compared to a reference sequenceusing one of the alignment programs described using standard parameters.One of skill in the art will recognize that these values can beappropriately adjusted to determine corresponding identity of proteinsencoded by two nucleotide sequences by taking into account codondegeneracy, amino acid similarity, reading frame positioning, and thelike. Substantial identity of amino acid sequences for these purposesnormally means sequence identity of at least 60%, more preferably atleast 70%, 80%, 90%, and most preferably at least 95%.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength and pH. However, stringent conditions encompasstemperatures in the range of about 1° C. to about 20° C. lower than theT_(m), depending upon the desired degree of stringency as otherwisequalified herein. Nucleic acids that do not hybridize to each otherunder stringent conditions are still substantially identical if thepolypeptides they encode are substantially identical. This may occur,e.g., when a copy of a nucleic acid is created using the maximum codondegeneracy permitted by the genetic code. One indication that twonucleic acid sequences are substantially identical is when thepolypeptide encoded by the first nucleic acid is immunologically crossreactive with the polypeptide encoded by the second nucleic acid.

(e)(ii) The term “substantial identity” in the context of a peptideindicates that a peptide comprises a sequence with at least 70% sequenceidentity to a reference sequence, preferably 80%, more preferably 85%,most preferably at least 90% or 95% sequence identity to the referencesequence over a specified comparison window. Preferably, optimalalignment is conducted using the homology alignment algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48:443-453. An indication thattwo peptide sequences are substantially identical is that one peptide isimmunologically reactive with antibodies raised against the secondpeptide. Thus, a peptide is substantially identical to a second peptide,for example, where the two peptides differ only by a conservativesubstitution. Peptides that are “substantially similar” share sequencesas noted above except that residue positions that are not identical maydiffer by conservative amino acid changes.

For example, an entire promoter sequence or nucleotide sequence ofinterest disclosed herein, or one or more portions thereof, may be usedas a probe capable of specifically hybridizing to corresponding promotersequences. To achieve specific hybridization under a variety ofconditions, such probes include sequences that are unique among thenucleotide sequences of interest or the promoter sequences and arepreferably at least about 10 nucleotides in length, and most preferablyat least about 20 nucleotides in length. Such probes may be used toamplify corresponding sequences from a chosen animal by PCR. Thistechnique may be used to isolate additional sequences from a desiredanimal or as a diagnostic assay to determine the presence of thesequences in an animal or animal cell.

In a PCR approach, oligonucleotide primers can be designed for use inPCR reactions to amplify corresponding DNA sequences from cDNA orgenomic DNA extracted from any animal of interest. Methods for designingPCR primers and PCR cloning are generally known in the art and aredisclosed in Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods andApplications (Academic Press, New York); Innis and Gelfand, eds. (1995)PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds.(1999) PCR Methods Manual (Academic Press, New York). Known methods ofPCR include, but are not limited to, methods using paired primers,nested primers, single specific primers, degenerate primers,gene-specific primers, vector-specific primers, partially-mismatchedprimers, and the like.

In hybridization techniques, all or part of a known nucleotide sequenceis used as a probe that selectively hybridizes to other correspondingnucleotide sequences present in a population of cloned genomic DNAfragments or cDNA fragments (i.e., genomic or cDNA libraries) from achosen organism. The hybridization probes may be genomic DNA fragments,cDNA fragments, RNA fragments, or other oligonucleotides, and may belabeled with a detectable group such as ³²P, or any other detectablemarker. Thus, for example, probes for hybridization can be made bylabeling synthetic oligonucleotides based on the nucleotide sequences ofinterest. Methods for preparation of probes for hybridization and forconstruction of cDNA and genomic libraries are generally known in theart and are disclosed in Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.).

Hybridization techniques include hybridization screening of plated DNAlibraries (either plaques or colonies; see, for example, Sambrook et al.(1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold SpringHarbor Laboratory Press, Plainview, N.Y.).

Hybridization of such sequences may be carried out under stringentconditions. By “stringent conditions” or “stringent hybridizationconditions” is intended conditions under which a probe will hybridize toits target sequence to a detectably greater degree than to othersequences (e.g., at least 2-fold over background). Stringent conditionsare sequence-dependent and will be different in different circumstances.By controlling the stringency of the hybridization and/or washingconditions, target sequences that are 100% complementary to the probecan be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of similarity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length,preferably less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. Duration of hybridizationis generally less than about 24 hours, usually about 4 to about 12hours.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA—DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284:T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)-0.61 (% form)-500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization, and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with approximately 90% identity aresought, the T_(m) can be decreased 10° C. Generally, stringentconditions are selected to be about 5° C. lower than the thermal meltingpoint (T_(m)) for the specific sequence and its complement at a definedionic strength and pH. However, severely stringent conditions canutilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower than thethermal melting point (T_(m)); moderately stringent conditions canutilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower thanthe thermal melting point (T_(m)); low stringency conditions can utilizea hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe thermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution), it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen (1993)Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, N.Y.); and Ausubel et al., eds. (1995) Current Protocols inMolecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience,New York). See Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).Thus, isolated sequences that have midkine modulating activity orpromoter activity and which hybridize under stringent conditions to thesequences disclosed herein, or to fragments thereof, are encompassed bythe present invention. Such sequences will be at least 85%, 90%, 95% to98% homologous or more with the disclosed sequences. That is, thesequence identity of sequences may range, sharing at least 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity.

Midkine promoters disclosed in the invention may be isolated from anyanimal, including but not limited to, rat, hamster, human, rabbit,mouse, monkey, chimpanzee, dog, pig, goat, sheep, cat, and cow. It isrecognized that any gene of interest can be operably linked to apromoter of the invention.

As noted, the heterologous nucleotide sequence of interest operablylinked to a promoter may be an antisense sequence for a targeted gene(e.g. midkine or a nucleotide sequence encoding a midkine modulatingagent). Thus, with these promoters, antisense constructionscomplementary to at least a portion of the messenger RNA (mRNA) for atargeted nucleotide sequence interest can be constructed. Antisensenucleotides are constructed to hybridize with the corresponding mRNA.Modifications of the antisense sequences may be made as long as thesequences hybridize to and interfere with expression of thecorresponding mRNA. In this manner, antisense constructions having 70%,preferably 80%, more preferably 85% sequence identity to thecorresponding antisensed sequences may be used. Furthermore, portions ofthe antisense nucleotides may be used to disrupt the expression of thetarget gene. Generally, sequences of at least 50 nucleotides, 100nucleotides, 200 nucleotides, or greater may be used. Thus, atissue-preferred promoter sequence may be operably linked to antisenseDNA sequences to reduce or inhibit expression of native midkine in atissue of interest.

By “nucleotide sequence of interest” is intended a sequence that is notnaturally occurring with the promoter sequence. While this nucleotidesequence is heterologous to the promoter sequence, it may be homologous,or native, or heterologous, or foreign, to the animal host.

It is recognized that the promoters may be used with their native codingsequences to increase or decrease expression resulting in a change inphenotype in the transformed animal or subject.

A number of promoters can be used in the practice of the invention. Thepromoters can be selected based on the desired outcome. In variousembodiments of the invention, a midkine modulating agent is an isolatednucleic acid molecule comprising an expression cassette comprising apromoter operably linked to a nucleotide sequence of interest.

By “promoter” or “transcriptional initiation region” is intended aregulatory region of DNA usually comprising a TATA box capable ofdirecting RNA polymerase II to initiate RNA synthesis at the appropriatetranscription initiation site for a particular coding sequence. Apromoter may additionally comprise other recognition sequences generallypositioned upstream or 5′ to the TATA box, referred to as upstreampromoter elements, which influence the transcription initiation rate. Itis recognized that having identified the nucleotide sequences for thepromoter regions disclosed herein, it is within the state of the art toisolate and identify further regulatory elements in the 5′ untranslatedregion. Thus, the promoter regions disclosed herein are generallyfurther defined by comprising upstream regulatory elements such as thoseresponsible for tissue and temporal expression of the coding sequence,enhancers and the like. Such elements are typically linked via a 5′untranslated region, which may further modulate gene expression, to acoding region of interest. In the same manner, the promoter elementsthat enable expression in the desired tissue such as pulmonary-tissue orsmooth muscle cells can be identified, isolated, and used with othercore promoters to confirm tissue-preferred expression. For genes inwhich the 5′ untranslated region does not affect cell specificity,alternative sources of 5′ untranslated leaders may be used inconjunction with these promoter elements.

A number of promoters can be used in the practice of the invention. Thepromoters can be selected based on the desired outcome. Where low levelexpression is desired, weak promoters will be used. Generally, by “weakpromoter” is intended a promoter that drives expression of a codingsequence at a low level. By “low level” is intended at levels of about1/10,000 transcripts to about 1/100,000 transcripts to about 1/500,000transcripts; conversely, a strong promoter drives expression of a codingsequence at a high level, or at about 1/10 transcripts to about 1/100transcripts to about 1/1000 transcripts. Alternatively, it is recognizedthat weak promoters also encompasses promoters that are expressed inonly a few cells and not in others to give a total low level ofexpression. Where a promoter is expressed at unacceptably high levels,portions of the promoter sequence can be deleted or modified to decreaseexpression levels.

It is recognized that to increase transcription levels or to altertissue specificity, enhancers and/or tissue-preference elements may beutilized in combination with the nucleotide sequences of interest. Forexample, quantitative or tissue specificity upstream elements from otherpulmonary tissue-preferred or smooth muscle cell preferred promoters maybe combined with the promoter regions of the invention to augmenttissue-preferred transcription. The human surfactant protein C promoteris a pulmonary tissue preferred promoter (Perl et al. (2002) TransgenicRes. 11:21-29, herein incorporated by reference in its entirety). Themurine smooth muscle 22 α (SM22α) promoter is a smooth muscle cellpreferred promoter (U.S. Pat. Nos. 6,015,711 and 5,837,534, hereinincorporated by reference in their entirety).

In other embodiments, the coding region is operably linked to aninducible regulatory element or elements. A variety of induciblepromoter systems have been described in the literature and can be usedin the present invention. These include, but are not limited to,tetracycline-regulatable systems (WO 94/29442, WO 96/40892, WO 96/01313,U.S. application Ser. No. 10/613,728); hormone responsive systems,interferon-inducible systems, metal-inducible systems, andheat-inducible systems, (WO 93/20218); and ecdysone inducible systems.Some of these systems, including ecdysone inducible and tetracyclineinducible systems are commercially available from Invitrogen (Carlsbad,Calif) and Clontech (Palo Alto, Calif), respectively.

By “inducible” is intended that a chemical stimulus alters expression ofthe operably linked nucleotide sequence of interest by at least 1%, 5%,preferably 10%, 20%, more preferably 30%, 40%, 50%, 60%, 70%, 80%, 90%,99% or more. The difference may be an increase or decrease in expressionlevels. Methods for assaying expression levels are described elsewhereherein. The chemical stimulus may be administered or withdrawn. Variouschemical stimuli are known in the art. In an embodiment, the chemicalstimulus is tetracycline, or an analog thereof.

One of the most widely used conditional systems is the binary,tetracycline-based system, which has been widely used in both cells andanimals to reversibly induce expression by the addition or removal oftetracycline or its analogues (See Bujard (1999). J. Gene Med.1:372-374; Furth, et al. (1994). Proc. Natl. Acad. Sci. USA91:9302-9306; and Mansuy & Bujard (2000). Curr. Opin. Neurobiol.10:593-596, herein incorporated by reference in their entirety.).

Another class of promoter elements includes those which activatetranscription of an operably linked nucleotide sequence of interest inresponse to hypoxic conditions. These include promoter elementsregulated at least in part by hypoxia inducible factor-1. Hypoxiaresponse elements include, but are not limited to, the erythropoietinhypoxia response enhancer element (HREE1), the muscle pyruvate kinaseHRE; the β-enolase HRE; and endothelin-1 HRE element, and chimericnucleotide sequence comprising these sequences. See Bunn and Poynton(1996) Physiol. Rev. 76:839-885; Dachs and Stratford (1996) Br. J.Cancer 74:S126-S132; Guillemon and Krasnow (1997) Cell 89:9-12; Firth etal. (1994) Proc. Natl. Acad. Sci. 91:6496-6500; Jiang et al. (1997)Cancer Res. 57:5328-5335; U.S. Pat. No. 5,834,306) herein incorporatedby reference in their entirety.

In an embodiment, the invention provides methods of preferentiallymodulating midkine activity in a tissue preferred manner (for example,pulmonary tissue or smooth muscle cells). It is recognized that thenucleotide sequence of interest of a midkine modulating agent which isan isolated nucleic acid molecule comprising an expression cassettecomprising a nucleotide sequence of interest can be operably linked toany tissue preferred promoter. The tissue preferred promoter allowsexpression of the nucleotide sequence of interest in a tissue preferredmanner. Tissues of particular interest include pulmonary tissue, smoothmuscle cells, pulmonary smooth muscle cells, and pulmonary vascularsmooth muscle cells.

By “pulmonary tissue-preferred” is intended that expression of theheterologous sequence is most abundant in pulmonary tissue, while someexpression may occur in other tissue types, particularly in tissuesdevelopmentally related to pulmonary tissue. Pulmonary-preferredexpression of a heterologous nucleotide sequence of interest occurs atlevels at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or100% greater than expression of the nucleotide sequence of interest innon-pulmonary tissue. By “pulmonary vascular tissue-preferred” isintended that expression of the heterologous sequence is most abundantin pulmonary vascular tissue, while some expression may occur in othertissue types, particularly in tissues developmentally related topulmonary vascular tissue. Pulmonary vascular-preferred expression of aheterologous nucleotide sequence of interest occurs at levels at least1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greaterthan expression of the nucleotide sequence of interest in non-pulmonaryvascular tissue. In an embodiment, tissue-preferred expression of aheterologous nucleotide sequence natively expressed in other tissue maybe desired. Expression of a heterologous nucleotide sequence from atissue-preferred promoter may not impact expression of the nucleotidesequence operably linked to its native promoter in other tissues.

By “smooth muscle cell-preferred” is intended that expression of theheterologous sequence is most abundant in smooth muscle cells, whilesome expression may occur in other cell types. Smooth musclecell-preferred expression of a nucleotide sequence of interest occurs atlevels at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or100% greater than expression of the heterologous nucleotide sequence ofinterest in non-smooth muscle cells. In an embodiment, smooth musclecell-preferred expression of a heterologous nucleotide sequence nativelyexpressed in other tissue may be desired. Expression of a heterologousnucleotide sequence from a smooth muscle cell-preferred promoter may notimpact expression of the nucleotide sequence operably linked to itsnative promoter in other tissues.

The invention provides methods of preferentially modulating midkineactivity or expression levels in a tissue of interest. In an embodiment,a midkine modulating agent is administered to the tissue of interest. Inan embodiment the midkine modulating agent is an isolated nucleic acidmolecule comprising an expression cassette comprising a nucleotidesequence of interest operably linked to a tissue preferred promoter.

By “preferentially modulating pulmonary tissue midkine expressionlevels” is intended that the expression of midkine in a transgenic cell,pulmonary tissue of a transgenic animal of the invention, or pulmonarytissue of a subject administered a midkine modulating agent differs fromexpression levels in a non-pulmonary transgenic cell or pulmonary tissueof a non-transgenic animal or untreated subject by at least 1%, 5%,preferably 10%, 20%, more preferably 30%, 40%, 50%, 60%, 70%, 80%, 90%,99%, or more. The difference may be an increase or decrease inexpression levels.

In addition to control regions that promote transcription, expressionvectors may also include regions that modulate transcription, such asrepressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

Such expression cassettes will comprise a transcriptional initiationregion comprising a promoter nucleotide sequence operably linked to theheterologous nucleotide sequence whose expression is to be controlled bythe promoter. Such an expression cassette is provided with at least onerestriction site for insertion of the nucleotide sequence to be underthe transcriptional regulation of the regulatory regions. The expressioncassette may additionally contain selectable marker genes.

The expression cassette will include in the 5′-to-3′ direction oftranscription, a transcriptional and translational initiation region,and a heterologous nucleotide sequence of interest. In addition tocontaining sites for transcription initiation and control, expressioncassettes can also contain sequences necessary for transcriptiontermination and, in the transcribed region a ribosome binding site fortranslation. Other regulatory control elements for expression includeinitiation and termination codons as well as polyadenylation signals.The person of ordinary skill in the art would be aware of the numerousregulatory sequences that are useful in expression vectors. Suchregulatory sequences are described, for example, in Sambrook et al.(1989) Molecular Cloning: A Laboratory Manual 2nd. ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.).

The expression cassette comprising a promoter sequence of the presentinvention operably linked to a heterologous nucleotide sequence or apromoter operably linked to midkine may also contain at least oneadditional nucleotide sequence for a gene to be co-transformed into theorganism. Alternatively, the additional sequence(s) can be provided onanother expression cassette.

The regulatory sequences to which the polynucleotides described hereincan be operably linked include promoters for directing mRNAtranscription. These include, but are not limited to, the left promoterfrom bacteriophage λ, the lac, TRP, and TAC promoters from E. coli, theearly and late promoters from SV40, the CMV immediate early promoter,the adenovirus early and late promoters, and retrovirus long-terminalrepeats.

The isolated modified midkine promoter sequences of the presentinvention can be modified to provide assays for a range of expressionlevels of a compound of interest or a midkine modulating agent. Thus,less than the entire promoter regions may be utilized and the ability todrive expression retained. However, it is recognized that expressionlevels of mRNA may be altered and usually decreased with deletions ofportions of the promoter sequences. Generally, at least about 20nucleotides of an isolated promoter sequence will be used to driveexpression of a nucleotide sequence. Truncated forms of the midkinepromoter sequence may also be used to assay compounds for midkinemodulating activity, particularly midkine expression modulatingactivity.

The invention encompasses isolated or substantially purified nucleicacid or protein compositions. An “isolated” or substantially “purified”nucleic acid molecule, polypeptide, or biologically active portionthereof, is substantially free of other cellular material, or culturemedium when produced by recombinant techniques or substantially free ofchemical precursors or other chemicals when chemically synthesized.Preferably, an “isolated” nucleic acid molecule is free of sequences(preferably polypeptide encoding sequences) that naturally flank thenucleic acid (i.e., sequences located at the 5′ and 3′ ends of thenucleic acid) in the genomic DNA of the organism from which the nucleicacid is derived. For example, in various embodiments, the isolatednucleic acid molecule can contain less than about 5 kb, 4kb, 3 kb, 2 kb,1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank thenucleic acid molecule in genomic DNA of the cell from which the nucleicacid is derived.

The promoters for midkine genes from alternative species may generallybe isolated from the 5′ untranslated region flanking their respectivetranscription initiation sites. Methods for isolation of promoterregions are well known in the art. By “isolated” is intended that thepromoter sequences have been determined and can be extracted bymolecular techniques or synthesized by chemical means. In eitherinstance, the promoter is removed from at least one of its flankingsequences in its native state.

Where appropriate, the heterologous nucleotide sequence whose expressionis to be under the control of a promoter sequence and any additionalnucleotide sequence(s) may be optimized for increased expression in thetransformed animal. That is, these nucleotide sequences can besynthesized using species preferred codons for improved expression, suchas rabbit-preferred codons for improved expression in rabbits ormouse-preferred codons in mice. Methods are available in the art forsynthesizing species-preferred nucleotide sequences. See, for example,Wada et al. (1992) Nucleic Acids Res. 20 (Suppl.), 2111-2118; Butkus etal. (1998) Clin Exp Pharmacol Physiol Suppl. 25:S28-33; and Sambrook etal. (1989) Molecular Cloning: A Laboratory Manual 2nd. ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., herein incorporatedby reference.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequencesthat may be deleterious to gene expression. The G-C content of theheterologous nucleotide sequence may be adjusted to levels average for agiven cellular host, as calculated by reference to known genes expressedin the host cell. In an embodiment, the sequence is modified to avoidpredicted hairpin secondary mRNA structures. In an embodiment thesequence is modified to yield hairpin RNA structures for use in siRNA.

The expression cassettes may additionally contain 5′ leader sequences inthe expression cassette construct. Such leader sequences can act toenhance translation. Translation leaders are known in the art andinclude: picornavirus leaders, for example, EMCV leader(Encephalomyocarditis 5′ noncoding region) (Elroy-Stein et al. (1989)Proc. Nat. Acad. Sci. USA 86:6126-6130); potyvirus leaders, for example,TEV leader (Tobacco Etch Virus) (Allison et al. (1986)); MDMV leader(Maize Dwarf Mosaic Virus) (Virology 154:9-20); and human immunoglobulinheavy-chain binding protein (BiP) (Macejak et al (1991) Nature353:90-94). Other methods known to enhance translation and/or mRNAstability can also be utilized, for example, introns, and the like.

In those instances where it is desirable to have the expressed productof the heterologous nucleotide sequence directed to a particularorganelle, particularly the mitochondria, the nucleus, the endoplasmicreticulum, the Golgi apparatus; or secreted at the cell's surface orextracellularly; the expression cassette may further comprise a codingsequence for a transit peptide. Such transit peptides are well known inthe art and include, but are not limited to, the transit peptide for theacyl carrier protein, the small subunit of RUBISCO, and the like.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose; in vitro mutagenesis; primerrepair; restriction; annealing; substitutions, for example, transitionsand transversions; or any combination thereof may be involved.

Reporter genes or selectable marker genes may be included in theexpression cassettes. Examples of suitable reporter genes known in theart can be found in, for example, Ausubel et al. (2002) CurrentProtocols in Molecular Biology. John Wiley & Sons, New York, N.Y.,herein incorporated by reference. The reporter utilized in the inventioncan be any reporter known in the art including, but not limited to,luciferases, blue fluorescent proteins, green fluorescent proteins, CAT,GUS, β-galactosidases, and midkine.

Selectable marker genes for selection of transformed cells or tissuescan include genes that confer antibiotic resistance. Examples ofsuitable selectable marker genes include, but are not limited to, genesencoding resistance to neomycin (Schwartz et al (1991) Proc. Natl. Acad.Sci.88:10416-20); chloramphenicol (Herrera Estrella et al. (1983) EMBOJ. 2:987-992); methotrexate (Herrera Estrella et al. (1983) Nature303:209-213; Meijer et al. (1991) Plant Mol. Biol. 16:807-820);hygromycin (Waldron et al. (1985) Plant Mol. Biol. 5:103-108; Zhijian etal. (1995) Plant Science 108:219-227); streptomycin (Jones et al. (1987)Mol. Gen. Genet. 210:86-91); spectinomycin (Bretagne-Sangard et al.(1996) Transgenic Res. 5:131-137); bleomycin (Hille et al. (1990) PlantMol. Biol. 7:171-176); sulfonamide (Guerineau et al. (1990) Plant Mol.Biol. 15:127-136); puromycin (Abbate et al (2001) Biotechniques31:336-40; cytosine arabinoside (Eliopoulos et al. (2002) Gene Ther.9:452-462); 6-thioguanine (Tucker et al. (1997) Nucleic Acid Research25:3745-46).

Other genes that could serve utility in the recovery of transgenicevents but might not be required in the final product would include, butare not limited to, examples such as GUS (b-glucuronidase; Jefferson(1987) Plant Mol. Biol. Rep. 5:387); GFP (green fluorescence protein;Wang et al. (2001) Anim Biotechnol 12:101 -110; Chalfie et al. (1994)Science 263:802), BFP (blue fluorescence protein; Yang et al. (1998) J.Biol. Chem. 273:8212-6), CAT; and luciferase (Riggs et al. (1987)Nucleic Acid Res. 15 (19):8115; Luchrsen et al. (1992) Methods Enzymol.216: 397-414). These genes are also suitable as reporters when operablylinked to a midkine promoter or modified midkine promoter of theinvention.

Embodiments of the invention provide methods of screening for a midkinepathway abnormality, screening subjects for a pulmonary disorder, andidentifying a midkine modulating agent. In these embodiments, theinvention provides isolated nucleic acid molecules comprising anexpression cassette comprising a modified midkine promoter of theinvention operably linked to a reporter. In the embodiments, theisolated nucleic acid molecule comprising a modified midkine promoteroperably linked to a reporter is incubated with a test sample orcompound of interest. By test sample is intended a biological samplesuch as, but not limited to, a subject exhibiting a smooth muscle cellrelated disorder or pulmonary disorder, cells, cell lysates, cellularfractions, tissues, mucosa, and secretions. In an embodiment, anisolated nucleic acid molecule comprising a modified midkine promoteroperably linked to a reporter is transformed into a cell and incubationof the modified midkine promoter with the test sample occurs within thecell. In an embodiment, the isolated nucleic acid molecule is incubatedwith the test sample in vitro. Methods of assaying reporter expressionlevels are known in the art and described elsewhere herein. Reporterexpression levels are measured by any means known to one skilled in theart.

The invention provides methods of screening subjects for a pulmonarydisorder, a smooth muscle cell related disorder, a pulmonary smoothmuscle cell related disorder, and a pulmonary vascular smooth musclecell related disorder. In the methods an isolated nucleic acid moleculecomprising a modified midkine promoter operably linked to a reporter isincubated with a test sample obtained from a tissue of interest.Reporter expression levels are measured by any means known to oneskilled in the art.

Any of the regulatory or other sequences useful in expression vectorscan form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included.

Modulation of a midkine activity, smooth muscle cell development,pulmonary development, smooth muscle cell related disorder, pulmonarydisorder, a pulmonary smooth muscle cell related disorder, or apulmonary vascular smooth muscle cell related disorder can be achievedby providing expression of heterologous products or increased expressionof endogenous products in a tissue of interest. Alternatively, theresults can be achieved by providing for a reduction of expression ofone or more endogenous products, particularly enzymes and cofactors inthe tissue of interest. These changes result in a change in phenotype ofthe transgenic animal. For example, a nucleotide sequence of interestcan be used to preferentially express midkine in a tissue of interestand alter the midkine expression pattern. In an embodiment, thenucleotide sequence of interest encodes a polypeptide having a midkinemodulating activity that alters midkine expression (e.g. TTF-1 andHIF1-α). Alternatively, the promoter is operably linked to aheterologous nucleotide sequence of interest from which antisense mRNAcomplementary to the coding sequence of a polypeptide of interest can beproduced. The antisense RNA inhibits production of the protein orpolypeptide of interest. Alternatively, the promoter sequences of theinvention can be used to produce small interfering RNAs.

Products of the heterologous nucleotide sequence include structuralproteins, enzymes, cofactors, hormones, signaling proteins, and thelike.

Nucleotide sequences of interest disclosed in the present invention, aswell as variants and fragments thereof, are useful in the geneticmanipulation of any animal when operably linked with a promoter capableof initiating transcription in the animal. The midkine sequences areprovided in expression cassettes for expression in a transgenic animalof the invention. The cassette will include 5′ to 3′ regulatorysequences operably linked to a heterologous nucleotide sequence. By“operably linked” is intended the transcription of the nucleotidesequence of interest is under the influence of the promoter sequence. Inthis manner, promoter nucleotide sequences may be provided in expressioncassettes along with nucleotide sequences of interest for expression inthe animal of interest, more particularly for tissue-preferredexpression in the animal. In an embodiment, heterologous nucleotidesequences of interest suitable for use in the invention include midkinemodulating agents including, but not limited to, nucleotide sequencesencoding thyroid transcription factor-1 (TTF-1, SEQ ID NO:3), midkine(SEQ ID NO:1, SEQ ID NO:25), hypoxia inducing factor-1 (HIF1-α, SEQ IDNO:5), transcription factors, midkine promoter binding polypeptides,antisense nucleotide sequences, and small interfering RNA (siRNA)sequences. In another embodiment heterologous nucleotide sequencessuitable for use in the methods of the invention include nucleotidesequences encoding reporters or reporter genes. Reporters are describedelsewhere herein.

By “stably transformed” is intended that the nuclear genome of theanimal cell or the nuclear genome of at least one cell of the animal hasincorporated at least one copy of the transgene. By “transgene” isintended a nucleic acid molecule having a nucleotide sequence comprisingeither an expression cassette or a disruption cassette. A transgenicanimal of the invention comprises at least one stably transformed cellcomprising the nucleotide sequence of interest. In an embodiment, thegenome of a germ-line cell of a transgenic animal comprises thenucleotide sequence of interest. A transgenic cell, e.g. a smooth musclecell, is a cell comprising at least one expression cassette ordisruption cassette isolated from a transgenic animal of the invention.Transgenic tissue, e.g. pulmonary tissue and pulmonary smooth musclecell, is tissue comprising transgenic cells. Transgenic tissue isisolated from a transgenic animal of the invention.

Transgenic animals of the invention are useful for studying the functionof midkine and identifying and evaluating modulators of midkineactivity, smooth muscle cell development, pulmonary development, smoothmuscle cell disorders, pulmonary disorders and pulmonary smooth musclecell disorders. A transgenic animal is preferably a mammal, for examplea mouse, in which one or more of the cells of the animal include atransgene. A transgene is exogenous DNA which is integrated into thegenome of a cell from which a transgenic animal develops and whichremains in the genome of the mature animal in one or more cell types ortissues of the transgenic animal. A genetically engineered host cell canbe used to produce non-human transgenic animals.

Transgenic animals that exhibit altered midkine expression are useful toconduct assays that identify compounds that affect midkine function suchas, but not limited to, vascular remodeling. Assays to determinevascular remodeling are known in the art and include, but are notlimited to, immunohistochemical analysis and morphometric analysis. Thealtered midkine expression may result in altered susceptibility to asmooth muscle cell related disorder, pulmonary disorder, or pulmonarysmooth muscle cell related disorder.

Methods for generating transgenic animals via embryo manipulation andmicroinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866; 4,870,009; 4,873,191; 6,201,165 and in Nagy et al Ed.(2003) Manipulating the Mouse Embryo Cold Spring Harbor Press, ColdSpring Harbor, N.Y.), herein incorporated by reference in theirentirety.

Similar methods are used for production of other transgenic animals. Atransgenic animal can be produced by introducing nucleic acid into themale pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. A transgenic founder animal can beidentified based upon the presence of the transgene in its genome and/orexpression of transgenic mRNA in tissues or cells of the animals. Atransgenic founder animal can then be used to breed additional animalscarrying the transgene. Moreover, transgenic animals carrying atransgene can further be bred to other transgenic animals carrying othertransgenes. A transgenic animal also includes animals in which theentire animal or tissues in the animal have been produced using thehomologously recombinant host cells described herein. Methods forproviding transgenic rabbits are described in Marian et al. (1999) J.Clin. Invest. 104:1683-1692 and James et al. (2000) Circulation101:1715-1721, herein incorporated by reference in their entirety.

Other examples of transgenic animals include non-human primates, sheep,dogs, pigs, cows, goats, rats, rabbits, and hamsters.

In an embodiment, the invention provides a method of altering expressionof a heterologous midkine nucleotide sequence in an animal, particularlyof altering tissue preferred expression of the heterologous nucleotidesequence. In an embodiment the tissue preferred expression is pulmonarytissue preferred expression. In another embodiment, the tissue preferredexpression is smooth muscle cell preferred expression. In an embodimentthe heterologous nucleotide sequence is operably linked to a tissuepreferred promoter. An expression cassette comprising the tissuepreferred promoter operably linked to the heterologous nucleotidesequence is used to transform an animal. In an embodiment a tissuepreferred promoter is operably linked to a nucleotide sequence encodinga polypeptide that enhances expression from a second promoter. Thesecond promoter is operably linked to a nucleotide sequence encodingmidkine (SEQ ID NO:25). Animal transformation methods are known in theart and reviewed elsewhere herein. The method yields a stablytransformed transgenic animal exhibiting altered expression of aheterologous nucleotide sequence.

In another embodiment, transgenic non-human animals can be producedwhich contain selected systems, which allow for regulated expression ofthe transgene. Examples of such systems include, but are not limited to,the cre/loxP recombinase system, the FLP recombinase system, and thetetracycline based system described elsewhere herein. If a tetracyclinebased system is used to regulate expression of the transgene, animalscontaining transgenes encoding both a transactivator and a selectedprotein are required. Such animals can be provided through theconstruction of “double” transgenic animals, e.g., by mating twotransgenic animals, one containing a transgene encoding a selectedprotein and the other containing a transgene encoding a transactivator.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut et al. (1997)Nature 385:810-813 and PCT International Publication Nos. WO 97/07668and WO 97/07669. In brief, a cell, e.g., a somatic cell, from thetransgenic animal can be isolated and induced to exit the growth cycleand enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to a pseudopregnant femalefoster animal. The offspring born of this female foster animal will be aclone of the animal from which the cell, e.g., the somatic cell, isisolated.

In one embodiment, a transgenic animal cell can be a fertilized oocyteor embryonic stem cell that can be used to produce a transgenic animalcomprising at least one stably transformed expression cassettecomprising the heterologous nucleotide sequence. Alternatively, atransgenic cell can be a stem cell or other early tissue precursor thatgives rise to a specific subset of cells and can be used to producetransgenic tissues in an animal. See also Thomas et al., (1987) Cell51:503 for a description of homologous recombination vectors. The vectoris introduced into an embryonic stem cell line (e.g., byelectroporation) and cells in which the introduced gene has recombinedwith the genome are selected (see e.g., Li, E. et al. (1992) Cell69:915). The selected cells are then injected into a blastocyst of ananimal (e.g., a mouse) to form aggregation chimeras (see e.g., Bradley,A. in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach,E. J. Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). A chimeric embryocan then be implanted into a suitable pseudopregnant female fosteranimal and the embryo brought to term. Progeny harboring the recombinedDNA in their germ cells can be used to breed animals in which all cellsof the animal contain the recombined DNA by germ line transmission ofthe transgene. Methods for constructing homologous recombination vectorsand homologous recombinant animals are described further in Bradley, A.(1991) Current Opinion in Biotechnology 2:823-829 and in PCTInternational Publication Nos. WO 90/11354; WO 91/01140; and WO93/04169, herein incorporated by reference in their entirety.

Delivery vehicles suitable for incorporation of a polynucleotide forintroduction into a host cell include, but are not limited to, viralvectors and non-viral vectors (Verma and Somia (1997) Nature389:239-242).

A wide variety of non-viral vehicles for delivery of a polynucleotideare known in the art and are encompassed in the present invention. Anisolated nucleic acid molecule can be delivered to a cell as naked DNA(WO 97/40163). Alternatively, a polynucleotide can be delivered to acell associated in a variety of ways with a variety of substances (formsof delivery) including, but not limited to, cationic lipids;biocompatible polymers, including natural and synthetic polymers;lipoproteins; polypeptides; polysaccharides; lipopolysaccharides;artificial viral envelopes; metal particles; and bacteria. A deliveryvehicle can be a microparticle. Mixtures or conjugates of these varioussubstances can also be used as delivery vehicles. A polynucleotide canbe associated non-covalently or covalently with these forms of delivery.Liposomes can be targeted to a particular cell type, e.g., to a smoothmuscle cell or a pulmonary cell.

Viral vectors include, but are not limited to, DNA viral vectors such asthose based on adenoviruses, herpes simplex virus, poxvirus such asvaccinia virus, and parvoviruses, including adeno-associated virus; andRNA viral vectors, including but not limited to, the retroviral vectors.Retroviral vectors include murine leukemia virus, and lentiviruses suchas human immunodeficiency virus. See Naldini et al. (1996) Science272:263-267.

Non-viral delivery vehicles comprising a polynucleotide can beintroduced into host cells and/or target cells by any method known inthe art, such as transfection by the calcium phosphate co-precipitationtechnique; electroporation; electropermeablization; liposome-mediatedtransfection; ballistic transfection; biolistic processes includingmicroparticle bombardment, jet injection, and needle and syringeinjection, or by microinjection. Numerous methods of transfection areknown to the skilled worker in the field.

Viral delivery vectors can be introduced into cells by infection.Alternatively, viral vectors can be incorporated into any of thenon-viral delivery vectors described above for delivery into cells. Forexample, viral vectors can be mixed with cationic lipids (Hodgson andSolaiman (1996) Nature Biotechnol. 14:339-342); or lamellar liposomes(Wilson et al. (1977) Proc. Natl. Acad. Sci. 74:3471-3475; and Faller etal. (1984) J. Virol. 49:269-272).

For in vivo delivery, the vector can be introduced into an individual ororganism by any method known to the skilled artisan.

Transgenic animals that exhibit altered midkine expression of theheterologous nucleotide sequence are useful to conduct assays thatidentify compounds that modulate midkine activity, smooth muscle celldevelopment, pulmonary development, smooth muscle cell relateddisorders, pulmonary disorders, and pulmonary smooth muscle cell relateddisorders. The altered midkine expression may result in alteredsusceptibility to a pulmonary disorder or a smooth muscle cell relateddisorder.

Disorders of interest include, but are not limited to, pulmonarydisorders, smooth muscle cell related disorders, pulmonary smooth musclecell related disorders, and pulmonary vascular smooth muscle cellrelated disorders.

A “pulmonary disorder” is any disorder or condition involving pulmonarytissue. Pulmonary disorders include but are not limited to, pulmonaryhypertension, asthma, primary pulmonary hypertension, secondarypulmonary hypertension, pulmonary arterial hyperplasia, hypoxemia,airway hyperactivity, idiopathic pulmonary hypertension, hypertrophy,hyperplasia, pulmonary hypertension secondary to hypoxemia,bronchopulmonary dysplasia, heart disease associated with pulmonaryvascular remodeling, lung inflammation associated with pulmonaryvascular remodeling, persistent pulmonary hypertension of the newborn,Eisenmenger's complex, congenital anomalies; atelectasis; diseases ofvascular origin, such as pulmonary congestion and edema, includinghemodynamic pulmonary edema and edema caused by microvascular injury,adult respiratory distress syndrome (diffuse alveolar damage), pulmonaryembolism, hemorrhage, and infarction, and pulmonary hypertension andvascular sclerosis; chronic obstructive pulmonary disease, such asemphysema, chronic bronchitis, bronchial asthma, and bronchiectasis; corpulmonale associated severe pulmonary diseases, such as emphysema andcystic fibrosis; diffuse interstitial (infiltrative, restrictive)diseases, such as pneumoconioses, sarcoidosis, idiopathic pulmonaryfibrosis, desquamative interstitial pneumonitis, hypersensitivitypneumonitis, pulmonary eosinophilia (pulmonary infiltration witheosinophilia), Bronchiolitis obliterans-organizing pneumonia, diffusepulmonary hemorrhage syndromes, including Goodpasture syndrome,idiopathic pulmonary hemosiderosis and other hemorrhagic syndromes,pulmonary involvement in collagen vascular disorders, and pulmonaryalveolar proteinosis; bronchopulmonary dysplasia; complications oftherapies, such as drug-induced lung disease, radiation-induced lungdisease, and lung transplantation; tumors, such as bronchogeniccarcinoma, including paraneoplastic syndromes, bronchioloalveolarcarcinoma, neuroendocrine tumors, such as bronchial carcinoid,miscellaneous tumors, and metastatic tumors; pathologies of the pleura,including inflammatory pleural effusions, noninflammatory pleuraleffusions, pneumothorax, and pleural tumors, including solitary fibroustumors (pleural fibroma) and malignant mesothelioma. Secondary pulmonaryhypertension is caused by numerous diseases and conditions includingemphysema, bronchitis, scleroderma, CREST syndrome, systemic lupuserythematosus, ventricular septal defects, atrial septal defects,chronic pulmonary thromboembolism, HIV infection, liver disease,fenfluramine use and dexfenfluramine use.

Phenotypes associated with pulmonary disorders include, but are notlimited to, altered midkine expression levels, dyspnea on exertion,oxygenation levels, tricuspid regurgitation, right ventricular dilation,ventricular hypertrophy, and morphology.

Assays to assess pulmonary disorders or phenotypes associated withpulmonary disorders include, but are not limited to, two-dimensionalechocardiography, assessment of oxygenation, pulmonary function testing,high resolution computed tomography of the chest, ventilation-perfusionlung scanning, and cardiac catheterization, electrocardiogram, chestradiography, 2-D echocardiography with Doppler flow studies, arterialblood gas analysis, pulmonary angiography, EIAs, light microscopy,multiplex RT-PCR, positron emission tomography, MRI, pulmonaryultrasound, and hematoxylin and eosin staining.

A “smooth muscle cell related disorder” is any disorder or conditioninvolving smooth muscle cells including, but not limited to, smoothmuscle cell proliferation, smooth muscle cell differentiation, asthma,airway hyperactivity, idiopathic pulmonary hypertension, pulmonaryhypertension secondary to hypoxemia, and heart disease or lunginflammation that is associated with pulmonary vascular remodeling andpulmonary hypertension.

Phenotypes associated with smooth muscle cell related disorders include,but are not limited to, percent actinization, distal pulmonary vesselcounts, angiogenic activity, and midkine expression.

Assays to assess smooth muscle cell related disorders include but arenot limited to actin staining, morphometric analysis, and midkineexpression analysis.

A “pulmonary smooth muscle related disorder” is any disorder orcondition involving smooth muscle cells in pulmonary tissue including,but not limited to, smooth muscle cell proliferation, smooth muscle celldifferentiation, asthma, airway hyperactivity, idiopathic pulmonaryhypertension, pulmonary hypertension secondary to hypoxemia, heartdisease or lung inflammation that is associated with pulmonary vascularremodeling and pulmonary hypertension. Pulmonary smooth muscle relateddisorders and phenotypes associated with pulmonary smooth muscle relateddisorders may be assayed by any means known to one skilled in the art,including but not limited to, the smooth muscle cell related disorderassays and the pulmonary disorder assays.

In various embodiments, the invention provides methods of modulating apulmonary disorder, smooth muscle cell related disorder, a pulmonarysmooth cell related disorder, or a pulmonary vascular smooth muscle cellrelated disorder. The methods comprise the steps of identifying asubject exhibiting the disorder of interest and administering a midkinemodulating agent to the subject. In an embodiment a clinician identifiesa subject of interest based on a physical examination, a potentialsubject's description of the symptoms, or review of a description of thepotential subject's symptoms; or a potential subject correlates hissymptom or symptoms with a description of the symptoms associated with adisorder of interest. The midkine modulating agent may increase ordecrease the one or more symptoms associated with the disorder. In anembodiment, the midkine modulating agent is administered directly to thetissue of interest. In an embodiment, a cell in the tissue of interestis transformed with an isolated nucleic acid molecule comprising anexpression cassette comprising a promoter operably linked to anucleotide sequence of interest. Promoters and nucleotide sequences ofinterest are discussed elsewhere herein. In various aspects, the midkinemodulating agent functions by binding midkine, binding a midkinereceptor, or altering midkine expression levels; the invention is notlimited by the midkine modulating agents′ method of function. A midkinemodulating agent may function by altering HIF1-α expression, HIF-1α/βbinding, nuclear localization, or other HIF-1α activity.

By “subject” is intended a mammal, e.g., a human, or an experimental oranimal or disease model or mammalian tissue or mammalian cells. Suitablesubjects include mammals, particularly humans, exhibiting a pulmonarydisorder, a smooth muscle cell related disorder, or a pulmonary smoothmuscle cell related disorder; tissue obtained from a mammal exhibiting aexhibiting a pulmonary disorder, a smooth muscle cell related disorder,or a pulmonary smooth muscle cell related disorder; cells obtained froma mammal exhibiting a exhibiting a pulmonary disorder, a smooth musclecell related disorder, or a pulmonary smooth muscle cell relateddisorder; and cells cultured from a mammal exhibiting a exhibiting apulmonary disorder, a smooth muscle cell related disorder, or apulmonary smooth muscle cell related disorder. The subject can also be anon-human mammal such as, but not limited to, a horse, hamster, guineapig, mouse, rabbit, dog, pig, goat, cow, rat, monkey, chimpanzee, sheep,or other domestic animal.

By “altered susceptibility” is intended that a transgenic animal of theinvention differs from a non-transgenic animal in the extent to whichthe transgenic animal of the invention exhibits a pulmonary disorderassociated phenotype, smooth muscle cell disorder related phenotype, ora pulmonary smooth muscle cell related disorder phenotype. The disorderphenotype may present during any stage of development including, but notlimited to, embryonically, post-natally, in the adult, and as the animalnears end of lifespan. In an embodiment, the disorder phenotype may beinduced by external stimuli such as, but not limited to, diet, exercise,chemical treatment, or surgical procedure.

In an embodiment, a transgenic animal or cell of the invention may beused to identify midkine modulating agents. To identify midkinemodulating agent, multiple transgenic animals of the invention, e.g. atleast a first and second transgenic animal, are provided. The terms“first,” “experimental,” or “test” transgenic animal refer to atransgenic animal to which a compound of interest is administered. Theterms “second” or “control” transgenic animal refer to a transgenicanimal to which a placebo is administered. In an embodiment, the firstand second transgenic animals are clonal, age-matched, gender-matched,and subject to similar environmental conditions. In an embodiment, morethan one animal may be a first transgenic animal. In an embodiment morethan one animal may be a second transgenic animal.

After administration of either the compound of interest or the placebo,the first and second transgenic animals are incubated for a period oftime. The period of time will have a predetermined duration appropriateto analysis of the phenotype. Such durations include, but are notlimited to, 30 seconds; 1, 5, 10, 30, or 60 minutes; 8, 12, 24, 36,or48hours; 3, 4, 5, 6, or 7 days; 2, 3, or 4 weeks; 2, 3, 4, 5, 6, 7, 8,9, 10, 11, or 12 months; up to 3 years. Monitoring of a midkine activitymay occur continuously; at a single interval; or at multiple intervals,such as, but not limited to, hourly, daily, weekly, and monthly. Anymethod of assaying a midkine activity known in the art may be used tomonitor the effects of the compound of interest on a transgenic animalof the invention.

In an embodiment, the midkine promoter and the modified midkinepromoters of the invention are used to identify a midkine modulatingagent. An isolated nucleic acid molecule comprising an expressioncassette comprising a midkine promoter or a modified midkine promoteroperably linked to a reporter is provided. The isolated nucleic acidmolecule is incubated with a compound of interest, and the reporter isassayed. Suitable reporters are described elsewhere herein. The periodof time will have a predetermined duration appropriate to analysis ofthe phenotype. Such durations include, but are not limited to, 30seconds; 1, 5, 10, 30, or 60 minutes; 8, 12, 24, 36, or 48 hours; 3, 4,5, 6, or 7 days; 2, 3, or 4 weeks; 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12months; up to 3 years. Monitoring of a midkine activity may occurcontinuously; at a single interval; or at multiple intervals, such as,but not limited to, hourly, daily, weekly, and monthly. Any method ofassaying a reporter activity known in the art may be used to monitor theeffects of the compound of interest.

In an embodiment, a transgenic animal of the invention may be used toidentify pulmonary disorder modulating agents, smooth muscle cellrelated disorder modulating agents, pulmonary smooth muscle cell relateddisorder modulating agents, pulmonary development modulating agents,smooth muscle cell development modulating agents, and pulmonary smoothmuscle cell development modulating agents. A “disorder modulating agent”is a compound that modulates a phenotype associated with a disorder ofinterest. A “development modulating agent” is a compound that modulatesa phenotype associated with development of a tissue of interest.Modulation may be an increase or decrease in the phenotype. A modulatingagent will modulate a development or disorder associated phenotype by atleast 1%, 5%, preferably 10%, 20%, more preferably 30%, 40%, 50%, 60%,yet more preferably 70%, 80%, 90%, or 100% as compared to an untreatedor placebo treatment effect. Methods for assaying development anddisorder associated phenotypes are described elsewhere herein. Anymethod of assaying a development or disorder of interest associatedphenotype known in the art may be used to monitor the effects of thecompound of interest on a transgenic animal of the invention.

To identify modulating agents, multiple transgenic animals of theinvention, e.g. at least a first and second transgenic animal, areprovided. The terms “first,” “experimental,” or “test ” transgenicanimal refer to a transgenic animal to which a compound of interest isadministered. The terms “second” or “control” transgenic animal refer toa transgenic animal to which a placebo is administered. In anembodiment, the first and second transgenic animals are clonal,age-matched, gender-matched, and subject to similar environmentalconditions. In an embodiment, more than one animal may be a firsttransgenic animal. In an embodiment more than one animal may be a secondtransgenic animal.

After administration of either the compound of interest or the placebo,the first and second transgenic animals are incubated for a period oftime. Compounds of particular interest are midkine modulating agents.The period of time will have a predetermined duration appropriate toanalysis of the phenotype. Such durations include, but are not limitedto, 30 seconds; 1, 5, 10, 30, or 60 minutes; 8, 12, 24, 36, or 48 hours;3, 4, 5, 6, or 7 days; 2, 3, or 4 weeks; 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,or 12 months; up to3 years. Monitoring of a development or disorderassociated phenotype may occur continuously; at a single interval; or atmultiple intervals, such as, but not limited to, hourly, daily, weekly,and monthly. Any method of assaying a development or disorder associatedphenotype known in the art may be used to monitor the effects of thecompound of interest on a transgenic animal of the invention.

By “development associated phenotype” is intended any phenotypeassociated with the development of a cell or tissue of interest.Development associated phenotypes include, but are not limited to, cellsurface marker expression, proliferation, differentiation, cellularmorphology, tissue morphology, percent actinization, percentmuscularization, pulmonary arterial hyperplasia, α-smooth muscle actinexpression, platelet-endothelial cell adhesion molecule expression,vessel formation, expression of cell or tissue specific polypeptides,and air vessel formation.

Development associated phenotype assays include, but are not limited to,scanning electron microscopy, light microscopy, hematoxylin and eosinstaining, immunostaining, gross dissection, antibody reactivity,expression pattern analysis, in situ hybridization, FACS analysis, andconfocal microscopy.

By “smooth muscle cell development associated phenotype” is intended anyphenotype associated with the development of smooth muscle cellsincluding, but not limited to, differentiation, proliferation, cellularmorphology, percent actinization, percent muscularization, a-smoothmuscle actin expression, and expression of smooth muscle cell-specificmarkers (Glukhova et al (1991) Am. J. Physiol. 261:78-80; Frid et al(1992) Dev. Biol. 153:185-193; herein incorporated by reference).

By “pulmonary development associated phenotype” is intended anyphenotype associated with pulmonary tissue or pulmonary cells includingbut not limited to, cell surface marker expression, expression ofpulmonary specific markers, tissue morphology, proliferation,differentiation, cellular morphology, percent muscularization, percentvascularization, pulmonary arterial hyperplasia, vessel formation, andpulmonary phenotypes discussed elsewhere herein.

An embodiment of the invention provides methods of modulating smoothmuscle development, particularly pulmonary smooth muscle development.The method comprises the step of administering a midkine modulatingagent to a mammal. Modulation of smooth muscle development can beassayed by monitoring a development associated phenotype. In anembodiment, the smooth muscle development associated phenotype that isaltered is proliferation. In an embodiment, the smooth muscledevelopment associated phenotype that is altered is differentiation.Administration of a midkine modulating agent increases or decreases thedevelopment associated phenotype. By “proliferation” is intended amitotic increase in cell number or an increase in the average size ofthe cell. By “differentiation” is intended the progression of apluripotent cell to a cell type from which fewer cell types can emerge.

The term “administer” is used in its broadest sense and includes anymethod of introducing a compound into a transgenic animal of the presentinvention. This includes producing polypeptides or polynucleotides invivo as by transcription or translation in vivo of polynucleotides thathave been exogenously introduced into a subject. Thus, polypeptides ornucleic acids produced in the subject from the exogenous compositionsare encompassed in the term “administer.”

A “compound” comprises, but is not limited to, nucleic acid molecules,small interfering RNAs, peptides, polypeptides, small molecules,glycoproteins, chemokine receptor inhibitors, antisense nucleotidesequences, peptidomimetics, lipids, antibodies, receptor inhibitors,ligands, sterols, steroids, hormones, kinases, kinase inhibitors,agonists, antagonists, ion-channel modulators, diuretics, enzymes,enzyme inhibitors, carbohydrates, deaminases, deaminase inhibitors,G-proteins, G-protein receptor inhibitors, calcium channel modulators,hormone receptor modulators, alcohols, phosphatases, lactones,neurotransmitter inhibitors, angiotensin converting enzyme inhibitors,vasodilators, anticoagulants, neurotransmitter receptor modulators,negative inotropic agents, β-blockers, Ca²⁺ antagonists, anti-arrhythmiaagents, vasodilators, midkine promoter binding agents, transcriptionfactors, TTF-1, and HIF-1α. A compound may additionally comprise apharmaceutically acceptable carrier.

As used herein the language “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration. Theuse of such media and agents for pharmaceutically active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the active compound, such media can be used in thecompositions of the invention. Supplementary active compounds can alsobe incorporated into the compositions. A pharmaceutical composition ofthe invention is formulated to be compatible with its intended route ofadministration. Examples of routes of administration include parenteral,e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation),transdermal (topical), transmucosal, and rectal administration.Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a carboxypeptidase protein or anti-carboxypeptidaseantibody) in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For oral administration, the agent can be contained in entericforms to survive the stomach or further coated or mixed to be releasedin a particular region of the GI tract by known methods. For the purposeof oral therapeutic administration, the active compound can beincorporated with excipients and used in the form of tablets, troches,or capsules. Oral compositions can also be prepared using a fluidcarrier for use as a mouthwash, wherein the compound in the fluidcarrier is applied orally and swished and expectorated or swallowed.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser, whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer. Compounds may also be delivered with supplemental oxygenadministered to a subject.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. “Dosage unit form” as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Midkine modulating agents identified by the methods of this inventionmay be used in the treatment of human individuals. Embodiments of theinvention provide methods of treating a pulmonary disorder, a smoothmuscle cell related disorder, and a pulmonary smooth muscle cell relateddisorder. The methods comprise -administering a therapeuticallyeffective amount of a midkine modulating agent to a subject exhibiting adisorder of interest. In an embodiment at least one cell of a tissue ofinterest is transformed with an isolated nucleic acid moleculecomprising an expression cassette comprising a promoter operably linkedto a nucleotide sequence of interest. In an aspect, the promoter is atissue-preferred promoter. Midkine modulating agents useful in thetreatment of a disorder discussed herein are provided in therapeuticallyeffective amounts. By “therapeutically effective amounts” is intended anamount sufficient to modulate the desired response. Appropriatetherapeutically effective amounts vary depending upon the actual midkinemodulating agent utilized, the delivery mode, and the agent's mode ofaction.

The following examples are offered by way of illustration and notlimitation.

EXPERIMENTAL EXAMPLE 1 Tissue Preparation

Mice were sacrificed by an intraperitoneal injection of a ketamine,xylazine, and acepromazine cocktail and exsanguinated by severing theinferior vena cava and descending aorta. Midline sternotomies wereperformed. Lungs were inflation fixed with 4% paraformaldehyde in PBSand stored in identical fixative at 4° C. Tissues were washed in PBS,dehydrated in a series of alcohols, and embedded in paraffin. Blockswere sectioned into 5 μm sections using standard techniques. Prior toimmunohistochemical staining, the sections were deparaffinized andrehydrated.

EXAMPLE 2 Immunohistochemical Analysis of Midkine, Clara Cell SecretoryProtein (CCSP), and Platelet-Endothelial Cell Adhesion Molecule(PECAM)-1

Rehydrated tissue sections were treated with 3% hydrogen peroxide inmethanol for 15 minutes. The sections were incubated with 2.5% horseserum in 0.1% TBS for 2 hours at room temperature. The sections wereincubated with midkine, CCSP, or PECAM-1 primary antibody at 4° C.overnight at appropriate dilutions. Control sections were incubatedovernight at 4° C. in blocking serum alone. The midkine primary antibodywas an affinity-purified goat polyclonal antibody targeted to thecarboxy-terminus of midkine (Santa Cruz Biotechnology, Inc. Santa CruzCalif.). Additional commercially available midkine antibodies include,but are not limited to, AF-258-PB, Lot WE02 (R&D Systems Inc.). Theanti-CCSP antibody was a goat polyclonal antibody targeted torecombinant mouse CCSP. The PECAM-1 primary antibody was monoclonal ratanti-mouse platelet-endothelial cell adhesion molecule antibody obtainedfrom Pharmigen, San Diego, Calif.

After application of primary antibody, sections were developed with abiotinylated horse anti-goat secondary antibody and a Vector Elite ABCkit (Vector Laboratories; Burlingame Calif.). Development in NiDAB wasfollowed by incubation with Tris-cobalt and counterstaining with nuclearfast red.

The sections were then dehydrated in an increasing series of ethanols,washed in three changes of xylene, and mounted under coverslips withPermount®. Colocalization was determined by analyzing serial sections tocompare immunolabeling of the various polypeptides.

EXAMPLE 3 Immunohistochemical Analysis of Pro-surfactant Protein(ProSP-C) and T₁α

Rehydrated tissue sections were treated with 3% hydrogen peroxide inmethanol for 15 minutes. The sections were incubated with 4% goat serumin 0.1% TBS for 2 hours at room temperature. The sections were incubatedwith ProSP-C and T₁α primary antibody at 4° C. overnight at appropriatedilutions. Control sections were incubated overnight at 4° C. inblocking serum alone. The ProSP-C primary antibody was a rabbitpolyclonal antibody targeted to the first 35 residues of the aminoterminus of the human SPC pro protein (Chemi-Con, Inc. AB 34.28); thisantibody detects the proSP-C polypeptide in mouse lung tissue. The T₁αprimary antibody was monoclonal hamster anti-mouse T₁α antibody (TheDevelopmental Studies Hybridoma Bank, University of Iowa; see Farr etal. (1992) J. Histochem Cytochem 40:651-664, herein incorporated byreference in its entirety).

After application of primary antibody, sections were developed with abiotinylated goat anti-rabbit secondary antibody and a Vector Elite ABCkit (Vector Laboratories; Burlingame Calif.). Development in NiDAB wasfollowed by incubation with Tris-cobalt and counterstaining with nuclearfast red.

The sections were then dehydrated in an increasing series of ethanols,washed in three changes of xylene, and mounted under coverslips withPermount®. Colocalization was determined by analyzing serial sections tocompare immunolabeling of the various polypeptides. Results from suchexperiments are presented in FIGS. 6 and 9.

EXAMPLE 4 Immunohistochemical Analysis of α-smooth Muscle Actin (αSMA)and Caldesmon

Rehydrated tissue sections were treated with 3% hydrogen peroxide inmethanol for 15 minutes. Inmmunohistochemistry was performed as outlinedin the Mouse on Mouse (M.O.M.) monoclonal kit obtained from themanufacturer (Vector Laboratories, Burlingame Calif.). The sections wereincubated with blocking serum for one hour at room temperature. Thesections were incubated with αSMA or caldesmon primary antibody atappropriate dilutions for 30 minutes. Control sections were incubatedwith blocking serum alone.

After application of primary antibody, sections were developed with abiotinylated mouse secondary antibody and a Vector Elite ABC kit (VectorLaboratories; Burlingame Calif.). Development in NiDAB was followed byincubation with Tris-cobalt and counterstaining with nuclear fast red.

The sections were then dehydrated in an increasing series of ethanols,washed in three changes of xylene, and mounted under coverslips withPermount®. Colocalization was determined by analyzing serial sections tocompare immunolabeling of the various polypeptides. Results from suchexperiments are presented in FIGS. 6 and 9.

EXAMPLE 5 Immunohistochemical Analysis of Midkine Expression in TTF-1Null Mice

TTF-1 null transgenic mice were bred and maintained as previouslydescribed (Kimura et al. (1996) Genes Dev. 10:60-69, Wert et al. (2002)Developmental Biol. 242:75-87; herein incorporated by reference in theirentirety). Sections of the TTF-1 null mice were prepared as describedelsewhere herein. Immunohistochemical analysis of midkine expression wasperformed on the sections as described elsewhere herein. Results fromsuch an experiment are presented in FIG. 1.

EXAMPLE 6 Northern Blot Analysis of Midkine Expression

Total RNA was isolated from lung tissue using Trizol® Reagent andaccording to the manufacturer's recommended protocol (Invitrogen,Carlsbad Calif.). RNA was stored at −80° C. until further analysis.Northern blot analyses were performed as described in Wikenheiser et al.(1993) Proc. Natl. Acad. Sci 90:11029-11033, herein incorporated byreference, with the following modifications. Ten micrograms of total RNAwas electrophoresed through 1% agarose/18% formaldehyde gels andtransferred to Hybond-N membranes (Amersham, Arlington Heights Ill). Themembranes were exposed to UV light.

α¹²P labeled DNA probes were generated using the Strip-EZ™ DNA kit(Ambion, Austin Tex.). The midkine probe was derived from the 881 basepair mouse cDNA (accession number:AA163237, SEQ ID NO:1) cloned into thepT3T7-Pac vector. The membranes were sequentially hybridized with α³²Plabeled DNA probes in hybridization buffer (0.5 M phosphate buffer, pH7.2, 3.5% SDS, 33% formamide, 1 mg/ml BSA, 1 mM EDTA, 20 mg/ml yeasttRNA) at 55° C. overnight. The Hybond-N membranes were washed in 2×SSC,0.1 % SDS at room temperature twice for 30 minutes, then in 0.2×SSC,0.1% SDS twice for one hour at 55° C. The blots were exposed to X-ARfilm with an intensifying screen and stored at −80° C. Results from onesuch experiment are presented in FIG. 5.

EXAMPLE 7 In Situ Hybridization of Midkine

Mouse midkine cDNA was incubated with EcoR1 and Not1 to yield an 881base pair fragment (SEQ ID NO:1). The EcoR1/Not1 fragment was ligatedinto the pGEM-11f(+) plasmid (Promega, Madison Wis.) using methods knownto one skilled in the art. ³⁵S-UTP labeled single-stranded sense andanti-sense RNA probes were prepared with a T7/SP6 Riboprobe® CombinationSystem and using the manufacturer's recommended protocol (Promega,Madison, Wis.). The labeled probes were precipitated with ethanol andresuspended in 100 nm dithiothreitol. The probes were diluted inhybridization solution (60% deionized formamide, 20 mM Tris-HCl, 5 mMEDTA pH 8.0, 0.3 M NaCl, 1× Denhardt's solution, 0.5 mg/ml yeast RNA,and 10% dextran sulfate).

In situ hybridization was performed according to the method of Wilkinsonand Green (1990) Postimplantation Mammalian Embryos (ed. A. J. Copp andD. L. Cockroft) London: Oxford University Press, pp. 155-171, hereinincorporated by reference in its entirety). Slides were immersed in 50%Ilford K5 nuclear track emulsion in 0.3 M ammonium acetate, dried, andexposed to autoradiography film for 7-21 days at 4° C. Hybridization wasvisualized after development of the slides with Kodak D19 developer at16° C. After development, sections were examined and photographed underphase optic and dark field illumination.

EXAMPLE 8 Identification and Isolation of the Murine Midkine Promoterand Truncated Modified Midkine Promoters

High fidelity polymerase chain reactions were performed with the ExpandLong Template PCR System (Roche, Indianapolis, Ind.). Oligonucleotideprimer sequences with Mlu1 restriction sites were designed. The sequenceof the upstream primer is set forth in SEQ ID NO:17. The sequence of thedownstream primer is set forth in SEQ ID NO:18. After an initialincubation at 94° C. for 1 minute, the reaction mixture underwent 35cycles of 20 seconds at 94° C., 30 seconds at 57° C., and 2 minutes at70°C. The amplified fragment was incubated with Mlul to yield a 2.5 kbfragment (SEQ ID NO:7). Subsequently the 2.5 kb fragment was ligatedinto the pGL3-basic luciferase reporter plasmid (Promega). The constructwas sequenced. Three truncated midkine promoter constructs (SEQ ID NO:8,SEQ ID NO:9, and SEQ ID NO:10) were generated with the appropriateupstream primers. The deletion promoters were sequenced also. Themodified promoters are diagrammed in FIG. 3.

EXAMPLE 9 Generation of Site Directed Modified Midkine Promoters

For generation of site directed mutations of the midkine promoter, the2.5 kb Midkine promoter pGL3 -basic reporter construct was used as atemplate. The site directed alterations were made using the QuickChange™Site-Directed Mutagenesis kit and following the manufacturer'srecommended protocol. Synthetic oligonucleotides comprising the desiredmutations were designed to replace six different regions of thepromoter. The modified midkine promoters set forth in SEQ ID NOS:11, 12,13, and 14 each contain a CAAG->CCCC alteration. The modified midkinepromoters set forth in SEQ ID NOS:15 and 16 each contain a RCGTG->CTTGCalteration.

To generate each modified promoter, the appropriate oligonucleotideswere used in a PCR reaction with the 2.5 kb midkine promoter pGL3 -basicreporter plasmid. The products of the PCR reaction were digested withDpnl. The nicked DNA was then transformed into XL1-blue supercompetentcells. Plasmids were isolated, and the modified promoter constructs wereconfirmed by sequencing. The modified promoters are diagrammed in FIGS.4 and 11.

EXAMPLE 10 Evaluation of TTF-1 and Midkine and Modified Midkine PromoterActivity

The JEG-3 cell line is a transformed human placental cell line withreduced TTF-1 expression. H-441 is a human epithelial cell line from apulmonary adenocarcinoma that expresses surfactant protein B (SP-B) andTTF-1. JEG-3 and H-441 cells were grown to 40-50% confluence in 35 mMtissue culture dishes. The cells were transfected with 3 plasmids at thefollowing concentrations: 500 ng/μl pRSV-βGal, 100 ng/μl MK-pGL3basic,and 0, 100, 200, 300,400, or 500 ng/μl pCMV-TTF 1, and pcDNA controlvector to bring the total to 1.1 μg total DNA.

After the cells reached confluence, the plates were washed with coldPBS. The cells were lysed, and snap-frozen for several hours. The plateswere scraped and the material was centrifuged. The cleared supernatantwas used for both luciferase assays and β-galactosidase assays.

B-galactosidase assays were performed as previously described (Bohinskiet al. (1994) Mol. Cell Biol. 14:5671-5681, herein incorporated byreference in its entirety). Reporter assays were normalized fortransfection efficiency based on the β-galactosidase activity.Luciferase activity was determined on 10 μl of extract at roomtemperature in 100 μl luciferase reagent (Promega) for 10 seconds aftera 2 second delay in a Monolight 3010 luminometer. Results from thesetypes of experiments are presented in FIGS. 3 and 4.

EXAMPLE 11 Evaluation of HIF-1α on Midkine and Modified Midkine PromoterActivity

The JEG-3 cell line is a transformed human placental cell line withreduced TTF-1 expression. MFLM-4 is a mouse fetal lung mesenchymal cellline. JEG-3 and MFLM-4 cells were grown to 40-50% confluence in 35 mMtissue culture dishes. The cells were transfected with 3 plasmids at thefollowing concentrations: 500 ng/μl pRSV-βGal, 100 ng/μl 2.5 kbMK-pGL3basic or 1.7 kb MK-pGL3 basic, and 0, 80, 160, 320, or 480 ng/μlpCMV-HIF1-α, and pcDNA control vector to bring the total to 1.08 μgtotal DNA.

After the cells reached confluence, the plates were washed with coldPBS. The cells were lysed and snap-frozen for several hours. The plateswere scraped and the material was centrifuged. The cleared supernatantwas used for both luciferase assays and β-galactosidase assays.

B-galactosidase assays were performed as previously described (Bohinskiet al. (1994) Mol. Cell Biol. 14:5671-5681, herein incorporated byreference in its entirety). Reporter assays were normalized fortransfection efficiency based on the β-galactosidase activity.Luciferase activity was determined on 10 μl of extract at roomtemperature in 100 μl luciferase reagent (Promega) for 10 seconds aftera 2 second delay in a Monolight 3010 luminometer. Results from thesetypes of experiments are presented in FIG. 11.

EXAMPLE 12 Generation of Transgenic Mice with an Inducible, PulmonaryPreferred Promoter Onerably Linked to Midkine

A pulmonary tissue preferred promoter, the human surfactant protein-Cpromoter was operably linked to the reverse tetracycline transactivator(rtTA) gene (Perl et al. (2002) Transgenic Res. 11:21-29), hereinincorporated by reference in its entirety. SP-C-rtTA mice wereidentified by PCR amplification as described in (Tichelaar et al. (2000)J. Biol. Chem. 275:11858-11864), herein incorporated by reference in itsentirety.

The (tetO)₇-CMV-MK was generated by inserting a 696 base pair mousemidkine cDNA (SEQ ID NO:25) downstream of a minimal CMV promotercontaining seven concatamerized tetracycline-receptor binding sites.Transgenic mice were generated by pronuclear injection using standardtechniques. Genotypes of transgenic mice were assayed by PCRamplification of a 559 base pair sequence of the transgene with theprimers set forth SEQ ID NO:19 and SEQ ID NO:20.

The SP-C-rtTA^(+/tg) mice and (tetO)₇-CMV-MK^(+/tg or tg/tg) mice werebred to yield double transgenic mice (SP-C-rtTA^(+/tg),(tetO)₇-CMV-MK^(+/tg or tg/tg)). Transgenic animals were fed doxycyclinecontaining food (625 mg/kg, Harlan Teklad, Madison Wis.).

Transgenic and control mice were housed and maintained under pathogenfree conditions in accordance with institutional guidelines. Embryonicday (E) 0 was determined to be the day when formation of a vaginal plugwas observed.

EXAMPLE 13 RT-PCR Analysis of Midkine Expression in Transgenic Mice

Lungs were collected from wild-type and double transgenic mice. TotalRNA was extracted from the lungs with Trizol® Reagent (Invitrogen) andaccording to the manufacturer's recommended protocol. Lung RNA wasresuspended and quantified. Aliquots of 2 μg total RNA were incubatedwith 1 μl RNAsin (Promega) and DnaseI (Invitrogen) at room temperaturefor 15 minuts. The reactions were incubated at 90° C. for 10 minutes.Reverse transcription reactions were performed with the SuperScrip™II-RT (Invitrogen) according to the manufacturer's recommended protocol.

PCR reactions were performed using 2 μl aliquots of the cDNA and Taqpolymerase (Roche). Products were run on a 1.5% agarose gel with theappropriate ladder standards. The nucleotide sequences of the primersused to amplify midkine are set forth in SEQ ID NO:21 and SEQ ID NO:22.The nucleotide sequences of the primers used to amplify HIF-1α are setforth in SEQ ID NO:23 and SEQ ID NO:24. The PCR reactions to amplify themidkine fragment underwent 35 cycles of incubation at 94° C. for 30seconds, 50° C. for 30 seconds, and 72° C. for 45 seconds. The PCRreactions to amplify the HIF-1α fragment underwent 30 cycles ofincubation at 94° C. for 30 seconds, 60° C. for 30 seconds, and 72° C.for 1 minute. For comparison, each experiment included two controlreactions: one lacking mRNA and one lacking reverse transcriptase.Results obtained from such an analysis are presented in FIG. 10. Detailsof midkine RT-PCR reactions are described in Reynolds, et al (2004). JBiol Chem. 279: 37124-37132 and Reynolds, et al (2003) Dev Dyn. 227:227-237, herein incorporated by reference in their entirety.

EXAMPLE 14 Morphometric Analysis of Right Ventricle Hypertrophy

Mice were sacrificed as described elsewhere herein. Hearts were excisedand stored in PBS at 4° C. The total heart mass was determined. Theright ventricle (RV) free wall was separated from the left ventricle(LV) and septum (S). The masses of the right ventricle, left ventricle,and septum were determined on an analytical balance. RV hypertrophy(RVH) was mathematically determined based on a ratio of RV/LV+S andRV/full body weight (FBW). Results obtained from such an analysis arepresented in FIG. 7.

EXAMPLE 15 Assessment of Distal Pulmonary Vessel Count

Transverse sections of mouse lungs were obtained as described elsewhereherein. The sections were immunostained with PECAM as describedelsewhere herein. The analyzer was blinded to the mouse origin of thetissue sections. Three fields in each section of the distal lung werechosen randomly. Vessels (15-50 μm external diameter) associated withalveoli were counted in the distal lung airspaces. Distal lung vesselsare arteries associated with alveolar ducts or walls. An average valuewas calculated. Results from such an analysis are presented in FIG. 7.

EXAMPLE 16 Assessment of Smooth Muscle Cell Induction

Lung sections were immunohistochemically stained with anti-a-SMAmonoclonal antibody (Sigma) as described above. The sections werecounter-stained with hematoxylin. Vessels with 15-50 μm externaldiameters associated with distal lung alveolar ducts or walls wereidentified. The extent of actin staining on each vessel was determined.Vessels with actin staining greater than 75% of the circumference werecharacterized as fully muscularized. Vessels with actin staining 25-50%of the circumference were characterized as partially muscularized.Vessels with actin staining less than 25% of the circumference werecharacterized as not muscularized. Results from such an analysis arepresented in FIG. 7.

EXAMPLE 17 Assessment of Hypoxia Exposure Effects in Vivo

Double transgenic and wild-type animals were placed in a hypobaricchamber and exposed to 10% O₂ for five weeks. The O₂ levels (10%) weredecreased from normoxic levels (20%) by displacement with N₂. Thechamber was furnished with regular changes of activated carbon (FisherScientific), Drierite (Hammond Drierite Co. LTD., Xenia, Ohio), andBaralyme (Allied Health Care Products, Inc. St. Louis. Mo.). Mice wereexposed to 12 hour light and 12 hour dark cycles and were allowed freeaccess to doxycycline containing food and water. Hypoxic conditions wererelieved briefly each day to weigh mice, clean cages, and replenish thefood and water.

EXAMPLE 18 Evaluation of HIF-1α on Midkine and Modified Midkine PromoterActivity

The JEG-3 cell line is a transformed human placental cell line withreduced TTF-1 expression. JEG-3 cells were grown to 40-50% confluence in35 mM tissue culture dishes. The cells were transfected with 3 plasmidsat the following concentrations: 500 ng/μl pRSV-βGal, 100 ng/μl 2.5 kbMK-pGL3basic, 100 ng/μl 1.7 kb MK-pGL3 basic, 100 ng/μl p2.5MK(Δ5′HRE)-luc, or 100 ng/μl p2.5 MK(Δ3′HRE)-luc, and 0, 100, or 200ng/μl pCMV-HIF1-α, and 0, 100, or 200 ng/μl pCMV-TTF-1, and pcDNAcontrol vector to bring the total to 1.08 μg total DNA.

After the cells reached confluence, the plates were washed with coldPBS. The cells were lysed and snap-frozen for several hours. The plateswere scraped and the material was centrifuged. The cleared supernatantwas used for both luciferase assays and β-galactosidase assays.

B-galactosidase assays were performed as previously described (Bohinskiet al. (1994) Mol. Cell Biol. 14:5671-5681, herein incorporated byreference in its entirety). Reporter assays were normalized fortransfection efficiency based on the β-galactosidase activity.Luciferase activity was determined on 10 μl of extract at roomtemperature in 100 μl luciferase reagent (Promega) for 10 seconds aftera 2 second delay in a Monolight 3010 luminometer. Results from thesetypes of experiments are presented in FIG. 11.

EXAMPLE 19 Assessment of Hypoxia Exposure Effects in Vitro

JEG-3 and MFLM-4 cells were maintained in Modified Eagle's Medium (MEM)and Dulbecco's modified Eagle's medium (DMEM), respectively. The mediawere supplemented with 10% Fetal Calf Serum, 2 mM glutamine, andantibiotics. Cells were routinely cultured in 5% CO₂, 95% air (normoxicconditions) at 37° C. At 80-90% confluence, the cells were divided andplated in 35 mm dishes. The freshly plated cells were grown in normoxicconditions for 18 hours. Half the cultures were then placed into anair-tight chamber (Thermo Forma Series II Water Jacketed CO₂ Incubator,Marietta Ohio) infused with a mixture of 5% CO₂, 5% O₂, and 90% N₂ for 4hours. Cells were then harvested and lysed. Total RNA was collectedusing the Absolutely RNA® RT-PCR Miniprep Kit (Stratagene) according tothe manufacturer's recommended protocol.

EXAMPLE 20 Pulmonary Vascular Remodeling Assessment

In order to assess the effect of midkine on pulmonary vasculature,doxycycline was provided to double transgenic and non-transgeniclittermates from conception to post-natal day 21 or from post-natal day21 to until sacrifice at week 9. Lung tissue sections were prepared asdescribed elsewhere herein. The tissue sections were immunostained forα-SMA as described above herein.

EXAMPLE 21 Pulmonary Arteriograms, Histology, and Arterial DensityQuantification

Adult mice were sacrificed with a 26% sodium pentobarbital euthanasiasolution and lungs were infused with a heated solution of gelatin andbarium through the pulmonary artery using methods known in the art(LeCras et al. (2003) Am. J. Physiol. 285:L1046-L1054, hereinincorporated by reference). Pulmonary arterial architecture was imagedby X-ray radiography. The left lungs were subsequently embedded inparaffin and sectioned as described elsewhere herein. Barium-filledpulmonary arteries were counted by a blinded observer in randomlyselected high-powered (60×) fields of distal lung. Fields containinglarge airways and/or large vessels were excluded.

EXAMPLE 22 Real-Time RT-PCR

Total RNA was isolated from mouse lungs and reverse transcribed to cDNA.Oligonucleotide SYBR green primer pairs for myocardin and β-actin weregenerated. Quantitative fluorogenic amplification of cDNA was performedin the Smart Cycler Processing Block, Model #SC1000-1 and using theLightCycler-DNA Master SYBR Green I Kit (available from Roche). Therelative abundance of mRNA was determined from standard curves generatedfrom the amplification from serially diluted standard pools of cDNA andnormalized to β-actin mRNA.

EXAMPLE 23 Midkine Expression in Human Subjects Exhibiting COPD

Pulmonary tissue was obtained from human subjects exhibitingCOPD/emphysema. The sections were immunostained with anti-midkineantibodies. Photomicrographs of one such experiment are presented inFIG. 8.

All publications, patents, and patent applications mentioned in thespecification are indicative of the level of those skilled in the art towhich this invention pertains. All publications, patents, and patentapplications are herein incorporated by reference to the same extent asif each individual publication or patent application were specificallyand individually incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1. A method of screening for a midkine transcription abnormality, saidmethod comprising: (a) providing an isolated nucleic acid moleculecomprising an expression cassette comprising a midkine promoter operablylinked to a nucleotide sequence encoding a reporter, wherein saidpromoter comprises a nucleotide sequence selected from the groupconsisting of: the nucleotide sequence as set forth in SEQ ID NO:7, SEQID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16; (b) incubating saidisolated nucleic acid molecule with a test sample in vitro; (c) assayingthe reporter by measuring the expression level or activity of thereporter; (d) comparing the expression level of the reporter operablylinked to said promoter in the presence of the test sample to theexpression level of said reporter operably linked to said promoter inthe presence of a control sample; and (e) identifying an alteredexpression level as a midkine transcription abnormality.
 2. The methodof claim 1, wherein said reporter is selected from the group consistingof: luciferases, blue fluorescent proteins, green fluorescent proteins,CAT, GUS, β-galactosidases, and midkine.
 3. The method of claim 1,wherein said isolated nucleic acid molecule is transformed into acultured cell.
 4. The method of claim 1, wherein incubating saidisolated nucleic acid molecule with a test sample occurs within acultured cell.
 5. The method of claim 1, wherein said test sample isselected from the group consisting of cell lysates and cellularfractions.
 6. A method of identifying a midkine modulating agent, saidmethod comprising: (a) providing an isolated nucleic acid moleculecomprising an expression cassette comprising a midkine promoter operablylinked to a nucleotide sequence encoding a reporter, wherein saidpromoter comprises a nucleotide sequence selected from the groupconsisting of: the nucleotide sequence as set forth in SEQ ID NO:7, SEQID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16; (b) incubating saidisolated nucleic acid molecule with a compound of interest in vitro; (c)expressing the isolated nucleic acid molecule in a sample selected fromthe group consisting of a cell, a cell lysate, a cellular fraction, anda tissue under in vitro conditions in the presence of the compound ofinterest; (d) assaying the reporter by measuring the expression level oractivity of the reporter; (e) comparing the expression level of thereporter operably linked to said promoter in the presence of saidcompound of interest to the expression level of said reporter operablylinked to said promoter in the absence of said compound of interest; and(f) identifying a compound of interest that modulates the expressionlevel of the reporter as a midkine modulating agent.
 7. The method ofclaim 1, wherein the control sample provides a baseline normalexpression level of the reporter.
 8. The method of claim 1, wherein saidpromoter comprises the nucleotide sequence as set forth in SEQ ID NO:7.9. The method of claim 1, wherein said promoter comprises the nucleotidesequence as set forth in SEQ ID NO:8.
 10. The method of claim 1, whereinsaid promoter comprises the nucleotide sequence as set forth in SEQ IDNO:9.
 11. The method of claim 1, wherein said promoter comprises thenucleotide sequence as set forth in SEQ ID NO:10.
 12. The method ofclaim 1, wherein said promoter comprises the nucleotide sequence as setforth in SEQ ID NO:11.
 13. The method of claim 1, wherein said promotercomprises the nucleotide sequence as set forth in SEQ ID NO:12.
 14. Themethod of claim 1, wherein said promoter comprises the nucleotidesequence as set forth in SEQ ID NO:13.
 15. The method of claim 1,wherein said promoter comprises the nucleotide sequence as set forth inSEQ ID NO:14.
 16. The method of claim 1, wherein said promoter comprisesthe nucleotide sequence as set forth in SEQ ID NO:15.
 17. The method ofclaim 1, wherein said promoter comprises the nucleotide sequence as setforth in SEQ ID NO:16.
 18. The method of claim 1, wherein the expressioncassette further comprises a selectable marker gene.
 19. The method ofclaim 1, wherein the expression level or activity of the reporter ismeasured with a luminometer.
 20. The method of claim 1, wherein the testsample comprises mucosa or a secretion.
 21. The method of claim 1,wherein the test sample is obtained from a subject exhibiting a smoothmuscle cell disorder.
 22. The method of claim 6, wherein said promotercomprises the nucleotide sequence as set forth in SEQ ID NO:7.
 23. Themethod of claim 6, wherein said promoter comprises the nucleotidesequence as set forth in SEQ ID NO:8.
 24. The method of claim 6, whereinsaid promoter comprises the nucleotide sequence as set forth in SEQ IDNO:9.
 25. The method of claim 6, wherein said promoter comprises thenucleotide sequence as set forth in SEQ ID NO:10.
 26. The method ofclaim 6, wherein said promoter comprises the nucleotide sequence as setforth in SEQ ID NO:11.
 27. The method of claim 6, wherein said promotercomprises the nucleotide sequence as set forth in SEQ ID NO:12.
 28. Themethod of claim 6, wherein said promoter comprises the nucleotidesequence as set forth in SEQ ID NO:13.
 29. The method of claim 6,wherein said promoter comprises the nucleotide sequence as set forth inSEQ ID NO:14.
 30. The method of claim 6, wherein said promoter comprisesthe nucleotide sequence as set forth in SEQ ID NO:15.
 31. The method ofclaim 6, wherein said promoter comprises the nucleotide sequence as setforth in SEQ ID NO:16.
 32. The method of claim 6, wherein the expressioncassette further comprises a selectable marker gene.
 33. The method ofclaim 6, wherein the compound comprises a nucleic acid molecule.
 34. Themethod of claim 6, wherein the compound comprises a peptide orpeptidomimetic.
 35. The method of claim 6, wherein the compoundcomprises a small molecule.
 36. The method of claim 6, wherein thesample comprises a cultured cell.
 37. The method of claim 6, wherein thesample comprises a cell lysate.
 38. The method of claim 36, wherein theexpression occurs within the cultured cell.
 39. The method of claim 6,wherein the expression level or activity of the reporter is measuredwith a luminometer.